117 Full

Physiol Rev
83: 117161, 2003; 10.1152/physrev.00018.2002.
Molecular Physiology of Low-Voltage-Activated

T-type Calcium Channels
EDWARD PEREZ-REYES
Department of Pharmacology, University of Virginia, Charlottesville, Virginia
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on October 20, 2016

I. Introduction 118
II. Electrophysiology of Native T-type Currents 120
A. Low-theshold calcium spikes 120
B. Neuronal 122
C. Heart 125
D. Kidney 127
E. Smooth muscle 128
F. Skeletal muscle 129
G. Sperm 129
H. Endocrine tissues 130
I. Cell lines 131
J. Single-channel recordings 131
III. Regulation 134
A. Hormonal inhibition 134
B. Hormonal stimulation 135
C. Guanine nucleotides 136
D. Protein kinases 136
E. Voltage 137
IV. Molecular Cloning of T-type Channels 138
A. Cloning 138
B. Structure 140
C. Distribution 141
D. Electrophysiology of recombinant channels 141
E. Auxiliary subunits? 146
V. Pharmacology 147
A. Antihypertensives 147
B. Antiepileptics 148
C. Anesthetics 149
D. Antipsychotics 149
VI. Conclusions 150
A. Physiological roles 150
B. Future directions 150
Perez-Reyes, Edward. Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels. Physiol Rev 83:
117161, 2003; 10.1152/physrev.00018.2002.T-type Ca2 channels were originally called low-voltage-activated
(LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neurons
Ca2 influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentials
mediated by Na channels. Burst firing is thought to play an important role in the synchronized activity of the
thalamus observed in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. In
addition to a pacemaker role, Ca2 entry via T-type channels can directly regulate intracellular Ca2 concentrations,
which is an important second messenger for a variety of cellular processes. Molecular cloning revealed the existence
of three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that found
in high-voltage-activated (HVA) Ca2 channels, and Na channels, indicating that they are evolutionarily related.
Hence, the 1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes are
expressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. The
electrophysiological activities of recombinant Cav3 channels are very similar to native T-type currents and can be
www.prv.org 0031-9333/03 $15.00 Copyright 2003 the American Physiological Society 117
118 EDWARD PEREZ-REYES
differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, and
smaller conductance of Ba2. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block by
Ni2. The goal of this review is to provide a comprehensive description of T-type currents, their distribution,
regulation, pharmacology, and cloning.
I. INTRODUCTION rapidly ( 1530 ms). The second type peaked at 0 mV

and inactivated slowly ( 2,000 ms). The existence of
Rises in intracellular calcium trigger a variety of pro- LVA currents was firmly established by the work of many
cesses including muscle contraction, chemotaxis, gene groups, including those headed by Kostyuk (115, 420),
expression, synaptic plasticity, and secretion of hor- Carbone and Lux (62), Armstrong (12), Bean (32), Feltz
mones and neurotransmitters. Although there are many (54), and Tsien (303, 306). Whole cell patch-clamp record-
channels and pumps involved in controlling intracellular ings showed that depolarizations in the 60 to 20 mV

Ca2 levels, voltage-gated Ca2 channels play a key role range elicited rapidly inactivating currents, while higher
in this process (50). Calcium is not only an important depolarizations elicited noninactivating currents. Plots of
second messenger, but its entry can also depolarize the the current versus test potential (I-V) showed two com-
plasma membrane, and thereby activate other voltage- ponents, with the LVA component appearing as a hump
gated ion channels. This property is especially important on the back of a larger HVA component. LVA currents
for neuronal T-type channels, which can generate low- also turned off, or deactivated, slower than HVA currents,
threshold spikes that lead to burst firing and oscillatory producing slow tail currents (62). This property was stud-
behavior (175, 377). This activity is especially prominent ied in great detail in GH3 cells, where slowly deactivating
in the thalamus, where it plays an important role in sen- (SD) tail currents were found to activate at lower thresh-
sory gating, sleep, and arousal (277). The term thalamo- olds than fast deactivating (FD) currents (12). Tsien and
cortical dysrhythmias has been coined to describe patho- colleagues (306) extended the classification of dorsal root
logical changes in these oscillations, and they have been ganglion (DRG) channels and proposed that these chan-
implicated in a wide range of neurological disorders in- nels be called T type for transient, L type for long lasting,
cluding absence epilepsy, the tremor associated with Par- and N type for neither T nor L type. LVA currents were
kinsons disease, tinnitus, neuropsychiatric disorders, and also found to be resistant to rundown, unlike HVA cur-
neurogenic pain (186, 242). rents that required cAMP, ATP, and Mg2 in the pipette
The ability of neurons to fire low-threshold Ca2 for stability (115). T- and L-type channels were also de-
spikes suggested the existence of low-voltage-activated scribed in cardiac myocytes from guinea pig ventricle and
(LVA) Ca2 channels. Since 1975 it has been recognized dog atrium and could be distinguished by their voltage
that there were at least two distinct types of Ca2 channel dependence, kinetics, single-channel currents, pharma-
(reviewed in Ref. 153). Hagiwara, Ozawa, and Sand, using cology, and conductance of Ca2 and Ba2 (32, 303). The
two-microelectrode voltage-clamp recordings from star- observation that T-type currents inactivate at lower mem-
fish eggs, found channels that were activated after small brane potentials than L-type currents led to the develop-
depolarizations of the membrane, LVA, and other chan- ment of an assay to separate these two components (32).
nels that required larger depolarizations of the membrane, The activity of both channels was recorded from a well-
high voltage activated (HVA) (154). LVA currents inacti- hyperpolarized potential such as 90 mV, then the activ-
vated faster, more completely, and at more negative mem- ity of L-type channels was recorded at a potential where
brane potentials than HVA currents. Similarly, LVA and T-type channels are inactivated and L type are not (typi-
HVA currents were described in the marine pileworm cally 50 mV), then the L-type currents are subtracted
Neanthes (123). Of historical note, the first recordings of from the T- plus L-type currents to isolate the T-type
mammalian LVA channels were made by Moolenar and current. Additional methods, such as tail current analysis,
Spector in 1978 (290), who used the two-microelectrode are required to isolate T-type currents in most other tis-
voltage-clamp technique on the mouse neuroblastoma sues due to the presence of HVA channels that also inac-
cell line N1E-115. However, these authors only observed tivate at 50 mV. Another possible contamination is volt-
one type of Ca2 channel, which in 10 mM Ca2 activated age-gated Na currents that appear to conduct Ca2
at 55 mV and peaked at 20 mV. Subsequent studies (ICa,TTX); however, these can be differentiated by their
have shown that N1E-115 cells provide a convenient sys- sensitivity to tetrodotoxin (TTX) (162). Hallmark features
tem to study the properties of native T-type currents (see of T-type channel electrophysiology are as follows: 1)
sect. III). Using quinidine to block K currents, Fishman they begin to open after small depolarizations of the
and Spector reported in 1981 (120) on the existence of plasma membrane (LVA); 2) their currents during a sus-
two types of mammalian Ca2 channel. One type acti- tained pulse are transient; 3) they close slowly upon
vated at 50 mV, peaked at 20 mV, and inactivated repolarization of the membrane, generating a SD tail cur-
Physiol Rev VOL 83 JANUARY 2003 www.prv.org

T-TYPE CALCIUM CHANNELS 119
rent; 4) they have a tiny, and equivalent, single-channel gests that gene duplication and divergence of an ances-
conductance of Ba2 and Ca2; 5) they are relatively tral Ca2 channel gene gave rise to LVA and HVA
insensitive to dihydropyridines; and 6) their steady-state subfamilies. Further duplication of the HVA gene gave
inactivation occurs over a similar voltage range as activa- rise to Cav1 and Cav2 subfamilies. This duplication
tion. In fact, T-type channels display a window current, must have occurred well over 500 million years ago,
i.e., there is a small range of voltages where T-type chan- since the nematode Caenorhabditis elegans contains
nels can open, but do not inactivate completely. This one member of each subclass. Eventually the Cav1
property may be particularly important in controlling in- subfamily evolved into four genes, while both the Cav2
tracellular Ca2 levels (45). In conclusion, studies on and Cav3 subfamilies evolved into three. Cloning and
native channels have provided a toolkit for separating expression studies have established that the Cav3 fam-
Ca2 channel subtypes, and these tools have proven use- ily encodes LVA T-type channels (sect. IV), while the
ful in the characterization of both native and recombinant Cav1 subfamily encodes L-type channels (although ex-
channels. pression of Cav1.4 has not been reported yet). These

T-type channels are expressed throughout the body, channels play a critical role in excitation-contraction
including nervous tissue, heart, kidney, smooth muscle, coupling in cardiac, skeletal, and smooth muscle. In
sperm, and many endocrine organs. These channels have fact, the Cav1.1 gene has evolved beyond a Ca2 chan-
been implicated in variety of physiological processes in- nel, and its physiological role in skeletal muscle is as a
cluding neuronal firing, hormone secretion, smooth mus- voltage sensor, coupling membrane depolarization di-
cle contraction, myoblast fusion, and fertilization. In gen- rectly to calcium release channels of the sarcoplasmic
eral, the electrophysiological properties of T-type reticulum (SR) (340). In contrast, cardiac and smooth
currents recorded from various cell types are similar, but muscle contraction requires influx of extracellular Ca2
differences have been noted in how they inactivate and in through Cav1.2 channels. In heart, this influx activates a
their pharmacology. This heterogeneity can be explained larger release of Ca2 from intracellular pools via a
in part by the existence of three T-type channels that are
mechanism called calcium-induced calcium release
encoded on separate genes (90, 228, 324).
(CICR) (37). Cav1.2 channels are an important thera-
Voltage-gated Ca2 channels have been classified
peutic target in the control of blood pressure and car-
by their electrophysiological and pharmacological
diac rhythm. The physiological roles of Cav1.3 channels
properties, and more recently by their amino acid se-
have been difficult to discern since pharmacological
quence identity. There are at least 10 genes encoding
tools have not been described that can separate its
1-subunits of voltage-gated Ca2 channels (Fig. 1).
activity from Cav1.2, and because these channels ap-
Alignment of their deduced amino acid sequences sug-
pear to be coexpressed in a number of tissues. In
contrast, the activity of Cav2 family members can be
selectively blocked by peptide toxins. Splice variation
of the Cav2.1 gene results in channels that differ in their
sensitivity to the spider toxin -Aga-IVA and therefore
encode both P- and Q-type channels (55). The Cav2.2
gene encodes N-type channels, which are characterized
by their irreversible and potent block by the snail toxin
-conotoxin-GVIA. Studies with these toxins indicate
that Cav2.1 and Cav2.2 channels mediate the Ca2 influx
into presynaptic terminals that trigger neurotransmitter
release. In contrast to these channels, it has been dif-
ficult to determine which native Ca2 current is carried
by Cav2.3 channels. Recombinant Cav2.3 channels re-
FIG. 1. Evolutionary tree of voltage-gated Ca
2
channels. Tree is semble T-type channels in their sensitivity to nickel and
based on an alignment of the putative membrane-spanning regions and their voltage dependence of inactivation, leading to the
pore loops of the human channels. Alignment of the full-length se-
quences yields a similar pattern, although all the branch points are
early suggestion that they might encode T-type chan-
shifted to the left due to inclusion of nonconserved sequences. Low- nels (376). However, Cav2.3 channels require stronger
voltage-activated (LVA) channels appear to have diverged from an an- depolarizations for channel opening, i.e., are HVA chan-
cestral Ca2 channel before the bifurcation of the high-voltage-activated
(HVA) channel family into Cav1 and Cav2 subfamilies. (Adapted from nels, and they deactivate 10-fold faster than T-type
Perez-Reyes E, Cribbs LL, Daud A, Yang J, Lacerda AE, Barclay J, channels (228). Studies with SNX-482, a tarantula toxin
Williamson MP, Fox M, Rees M, and Lee J-H. Molecular characterization that selectively blocks recombinant Cav2.3 channels,
of T-type calcium channels. In: Low Voltage-activated T-type Calcium
Channels, edited by Tsien RW, Clozel J-P, and Nargeot J. Chester, UK: suggests that these channels mediate R-type currents in
Adis International, 1998, p. 290 305.) some tissues (299).

Electrophysiological studies of recombinant chan- II. ELECTROPHYSIOLOGY OF NATIVE

nels show that Cav3.1 (formerly 1G) and Cav3.2 (1H) T-TYPE CURRENTS
have similar activation and inactivation kinetics, but can
be differentiated by their recovery from inactivation and
sensitivity to block by nickel (209, 230, 351). The kinetic A. Low-Threshold Calcium Spikes
properties of these 1-subunits closely resemble native
T-type channels. In contrast, recombinant Cav1 and Cav2 Low-threshold calcium spikes (LTS) have been de-
channels require auxiliary subunits for normal gating be- scribed in slices and in isolated neurons from a variety of
havior. This suggests that native T-type channels may be brain nuclei such as inferior olive (243, 244), thalamic
formed by a single 1-subunit. Cav3.3 (1I) channels also relay (96, 183), medial pontine reticular formation (146),
generate LVA currents; however, these currents activate lateral habenula (437), septum (10), deep cerebellar nu-
and inactivate much more slowly than T-type channels. clei (241), CA1-CA3 of the hippocampus (163, 307), asso-
Native currents with these properties have only been ciation cortex (122), paraventricular (399) and preoptic

described in a limited number of studies (19, 99, 177, 316, nuclei of the hypothalamus (385), dorsal raphe (60), glo-
398). Although there is no drug that is highly selective bus pallidus (296), and the subthalamic nucleus (40).
(10-fold) for T-type over other ion channels, block of Typically the spike is crowned by a burst of action poten-
T-type channels appears to contribute to the therapeutic tials (Fig. 2). Addition of TTX to block voltage-gated Na
usefulness of antihypertensives, antiepileptics, anesthet- channels abolishes the fast action potentials, effectively
ics, and possibly antipsychotics. Therefore, T-type chan- isolating the slowly activating and inactivating LTS. Nu-
nels provide a novel target for drug development. merous studies have shown that the LTS is mediated by a
2
FIG. 2. Low-threshold Ca spikes generate burst firing. A: many neurons can generate two distinct patterns of action
potential firing in response to a depolarizing stimulus. Regular, or tonic, firing is elicited when the neuron is depolarized
from a resting membrane potential near 55 mV. In contrast, when the membrane potential is below 70 mV, the same
depolarizing stimulus triggers a high-frequency burst of action potentials. [From Huguenard JR. Low-voltage-activated
(T-type) calcium-channel genes identified. Trends Neurosci 21: 451 452, 1998, with permission from Elsevier Science.]
B: a representative example of currents that generate burst firing. Depolarization of the plasma membrane by hyper-
polarization-activated current (Ih) leads to activation of T-type currents (IT), and a second phase of depolarization called
the low-threshold Ca2 spike. Riding on top of the low-threshold Ca2 spike are a burst of Na spikes mediated by fast
voltage-gated Na channels. High-threshold Ca2 and K currents can also be activated by the low-threshold calcium
spikes. Ca2 entry during the burst leads to activation of Ca2-activated K currents, which in combination with
voltage-gated K channels repolarize the membrane. (From Bal T and McCormick DA. Synchronized oscillations in the
inferior olive are controlled by the hyperpolarization-activated cation current Ih. J Neurophysiol 77: 31453156, 1997.)
C: thalamic neurons of transgenic mice lacking expression of Cav3.1 do not fire bursts. Current-clamp recordings are
from neurons held at 60, 70, or 80 mV, then depolarized or hyperpolarized by current injections of varying
magnitudes as indicated below the set of traces. When the resting membrane potential is 60 mV, a depolarizing stimulus
triggers tonic firing in both wild-type (/) and transgenic (/) animals. When the membrane potential (Vm) is lowered
to 70 mV, a hyperpolarizing stimulus triggers burst firing in neurons from wild-type, but not transgenic animals. When
the resting membrane potential is 80 mV, a depolarizing stimulus only triggers burst firing in neurons from wild-type
animals. (From Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, and Shin HS. Lack of the burst firing
of thalamocortical relay neurons and resistance to absence seizures in mice lacking 1G T-type Ca2 channels. Neuron
31: 35 45, 2001, with permission from Elsevier Science.)

Ca2 conductance; it is not observed in Ca2-free external These Na-dependent action potentials are brief (12 ms)
solutions, and it can be blocked by divalent cations such and of high amplitude (80 mV), and in some thalamic
as Co2 and Ni2. Definitive proof that thalamic LTS are neurons of very high frequency (400 Hz). High-threshold
mediated by T-type channels was provided by studies on Ca2 channels will also begin to open at potentials more
transgenic mice, where knockout of the Cav3.1 gene abol- positive than 40 mV (385). This will lead to more influx
ished these spikes and burst firing (Fig. 2C) (206). of Ca2 and, if present, activation of Ca2-dependent K
With a few notable exceptions (7, 191), LTS cannot channels, which can lead to an afterhyperpolarization
be triggered by depolarization of the neuron from the (AHP) (40, 244). Low-threshold channels can recover
resting membrane potential, which is typically between from inactivation during the AHP and could begin to fire
60 and 65 mV. However, LTS can be observed after a again as the membrane potential returns to its resting
hyperpolarizing pulse is delivered. This process has been level. Hyperpolarizations can also activate Ih channels,
called deinactivation and is due to recovery of channels which allow the influx of both Na and K, thereby depo-
from inactivation. Table 1 lists a number of studies where larizing the cell and directly triggering another LTS (Fig. 2).

the voltage and time dependence for recovery of these In this scenario Ih are the primary pacemaker current (254).
spikes has been examined in detail. LTS began to appear Clearly there are a variety of ionic conductances that medi-
after the neuronal membrane was hyperpolarized below ate pacemaker activity, and it is an oversimplification to
69 mV, and full-amplitude spikes were observed when implicate a single channel as the mediator of a pacemaker
the membrane was below 73 mV. The rate of recovery current. This is especially true in the heart (61).
was measured by varying the duration of a hyperpolariz- Another way that low-threshold channels are in-
ing pulse, then testing for the presence of an LTS upon volved in generating oscillations includes reciprocal con-
return to the resting membrane potential. These studies nections with an inhibitory interneuron. Due to its impor-
found that the LTS deinactivated relatively quickly, with tant role in controlling brain activity, the best studied of
half of it recovering in 100 ms and full recovery after 200 these circuits involves the GABAergic neurons of the
ms. Therefore, one physiological role of the LTS is as a thalamic reticular nucleus that regulate the activity of
pacemaker. Although increases in intracellular concentra- thalamic relay neurons (377). Interestingly, the firing pat-
tions of Ca2 act as a second messenger for a variety of terns of thalamic relay neurons vary with the state of
processes, in this case the important property is that consciousness (277, 378). In the awake state and during
influx of the divalent cation leads to direct depolarization rapid-eye-movement sleep, they fire in tonic mode: the
of the membrane. The amplitude of this depolarization resting membrane potential is relatively depolarized, the
was typically 25 mV (Table 1), which raised the mem- LTS is inactivated, and excitatory inputs faithfully trigger
brane potential to approximately 40 mV. Since the action potentials. In deep sleep, or during thalamocortical
threshold of many voltage-gated Na channels is around dysrhythmias, these neurons fire in an oscillatory mode
55 mV, the LTS can trigger a burst of action potentials. called burst firing; the resting membrane potential is hy-
TABLE 1. Properties of neuronal low-threshold Ca2 spikes
Resting Deinact V Deinact V Deinact Time Deinact Time

Membrane Threshold, Amplitude, Threshold, Midpt, Midpt, Max, Reference
Brain Region Potential, mV mV mV mV mV ms ms Nos.
Subthalamic nucleus 50 20 78 80 120 40

Septal nucleus 60 14 63 77 70 175 10
Hippocampus, CA1CA3 60 63 11 307
Thalamic nuclei 64 54 23 55 65 60 170 183
Lateral thalamic nuclei 60 65 20 69 50 96
Lateral geniculate nuclei 30 75 66 457
Lateral geniculate nuclei 60 68 35 64 72 456
Lateral habenular nucleus 62 65 30 65 68 437
Paraventricular nucleus 66 60 40 200 300 399
Medial preoptic nucleus 60 34 73 77 120 400 385
Dorsal raphe nucleus 67 60 70 400 800 60
Cerebellar nuclei 57 24 72 78 241
Cerebellar nuclei 58 65 20 72 77 200 400 2
Inferior olivary nucleus 64 65 35 70 75 40 75 243, 244
Hypoglossal motoneurons 60 65 10 83 200 400 421
The properties of low-threshold Ca2 spikes (LTS) were measured using current-clamp recordings. Threshold was defined by the voltage
where a depolarizing pulse triggers a LTS. The amplitude of the LTS is shown and does not include the fast Na-dependent action potential. The
voltage and time dependence for deinactivation (recovery) of the LTS spike is shown. Deinact., deinactivation; midpt, midpoint; CA1CA3,
hippocampal pyramidal neurons.

perpolarized, allowing full-amplitude low-threshold spik- of constant duration (isochronal), which assumes that the
ing (Fig. 2A). Such firing is thought to underlie spike-wave rates of channel activation and inactivation are voltage
discharges observed during absence seizures (176). Sim- independent. However, this is not the case, so this method
ilar patterns of brain activity have been detected in a can underestimate channel activation at negative poten-
variety of neurological disorders by magnetoencephalog- tials where channels open slowly. It can also underesti-
raphy and have been termed thalamocortical dysrhyth- mate activation if channels inactivate during the test
mias (242). pulse. This problem can be overcome by varying the
duration of the activating pulse, so that the tail current is
elicited at the peak of the current (288). So far this
B. Neuronal
method has only been applied to recombinant channels.
Another experimental variable that affects the position of
1. Isolated neurons
the activation curve is the concentration and choice of
LVA T-type currents have been characterized using charge carrier. Millimolar concentrations of positively

whole cell voltage-clamp recording of neurons isolated charged divalent cations can bind to surface charges on
from a variety of brain regions. Very large T-type currents both the channel and the membrane, thereby reducing the
(1 nA) have been recorded from cholinergic neurons of effective transmembrane voltage gradient (164). An effect
the basal forebrain (6), from relay neurons of the ventro- called surface charge screening. Increasing external
basal thalamus (222), and from sensory neurons of dorsal [Ca2] from 2 to 10 mM shifts the apparent gating of
root ganglia (436). Prominent T-type currents have been T-type channels by 10 mV (128).
found in neurons from many brain regions (Table 2). I-V A hallmark of T-type currents is that they are tran-
relationships are typically measured by holding the cell at sient; currents reach a peak then decay. In solutions
90 mV, and then currents are elicited by depolarizing where K channels are blocked, this decay is due to
pulses that increase in 10-mV increments. T-type currents channel inactivation. The inactivation rate is measured by
begin to activate when the membrane is depolarized fitting the current trace with an exponential function and
above 60 mV. At threshold potentials, the currents ac- is typically reported with a in milliseconds. Plots of
tivate relatively slowly, requiring many milliseconds to inactivation rate versus test potential reveal two phases:
reach peak, and also inactivate relatively slowly. At higher inactivation is slow at 60, gets faster between 50 and
potentials both activation and inactivation are faster. This 30, then is constant above 30 mV. Activation rates
produces a stereotypical pattern in the family of current have a similar voltage dependence, leading to the notion
traces where successive records cross each other, remi- that channels must activate before inactivating, and that
niscent of voltage-gated Na channels. In general, HVA the inactivation process is essentially voltage-indepen-
channels do not produce this criss-crossing pattern be- dent. Although most T-type currents inactivate relatively
cause inactivation rates are much slower than activation rapidly (30 ms), there are also reports of slowly inacti-
rates (334). I-V curves commonly show two components, vating currents (Table 2). This variability was interpreted
with the LVA currents peaking at 30 mV while the HVA as evidence for multiple T-type channel genes (179). This
currents peak around 0 mV. In many neurons, T-type hypothesis was supported by the cloning of T-type chan-
currents can be measured in isolation with test pulses to nels that differed in this property; Cav3.1 and 3.2 display
30 mV and below. However, at higher potentials both fast inactivation, while Cav3.3 displays slowly inactivating
LVA and HVA currents are activated, complicating esti- currents. Notably, Cav3.3 mRNA is expressed in the same
mates of the LVA activation curve. Both I-V curves show brain regions as the slow currents (177, 228, 393). Exclud-
a peak then decrease at more positive potentials, which is ing these cases, the average inactivation from 22 studies
due to a reduction of the electrochemical gradient for is 20 ms.
calcium ions as the test potential approaches the theoret- The voltage dependence of inactivation is also mea-
ical reversal potential (100 mV; see Fig. 7 for example). sured at steady-state by holding the membrane at vary-
Therefore, the fraction of channels activated by a test ing potentials (prepulse), then assaying for available T-
pulse continues to increase beyond the peak of the I-V type channels with a test pulse to 30 mV (h). To
curve. This explains why estimates of the midpoint of approximate steady-state, many studies have used a pre-
channel activation (V0.5) derived by normalizing the cur- pulse duration of 1 s, which is considerably longer than
rent at each test potential to the maximum current ob- the time required to inactivate open channels. However,
served (I/Imax) produces values that are more negative increasing the prepulse duration to 10 s results in a
(50 mV) than estimates that take into account changes 10-mV shift in the apparent h curve (53, 389). Despite
in driving force (41 mV). To circumvent this problem, the use of different prepulses and concentration of diva-
activation curves have also been estimated by measuring lent cations, there is good agreement between the 34
the amplitude of slowly deactivating tail currents at a studies shown in Table 2, and the average midpoint of the
constant voltage. Most of these studies used a test pulse h curve is 77 mV (Table 2). Somewhat surprisingly this

TABLE 2. Properties of T-type currents in isolated neurons

Divalent, Threshold, IV Peak, Imax, V0.5, h , inact, Reference
Tissue mM mV mV pA mV mV ms recov, ms Nickel, M Nos.
Basal forebrain
Striatum 5 Ca 60 40 90 53M 88 288 170
Accumbens 5 Ca 64 33 51 43C 80 397 80
Septum 2.5 Ca 54 1,320 40M 49 16 5 6
Septum 2 Ba 70 40 500 59M 84 147
Amygdala 10 Ca 60 20 20 38C 70 300 188
Cerebral cortex
Temporal 2 Ca 60 30 47 86 400 50 (28%) 354
Piriform 5 Ca 60 30 250 44T 86 22 257
Hippocampus
Dentate granule 2 Ca 50 30 224 65 25 458

LM 2 Ca 60 30 100 48M 84 12 100 400 126
interneurons
CA1 pyramidal 10 Ca 60 30 150 46C 79 15 430 (63%) 389
7,000
CA1, 3 pyramidal 2 Ba 57 100 76 25 820 400
Thalamus
Ventrobasal 3 Ca 60 25 350 52T 84 20 251 85
Ventrobasal 3 Ca 60 30 280 59T 81 30 350 200 (66%) 179
Ventrobasal 5 Ca 70 1,400 96 15 353 222
LGN 10 Ca 48 15 186 67 36 299 500 (83%) 387
LGN 5 Ca 60 23 200 45M 64 20 615 50 (100%) 159
LGN-interneuron 2 Ca 70 110 55M 81 20 218 318
LGN-relay 2 Ca 70 289 64M 86 20 206 318
Reticular 3 Ca 50 10 131 49T 78 80 570 200 (54%) 179
Reticular 2 Ca 70 50 128 64T 85 25 275 50 (38%) 409
Lateral habenula 1 Ca 65 35 150 62T 81 58W 507 177
Hypothalamus
Hypothalamus 10 Ca 65 30 93 40 940 600 4
Hypothalamus 10 Ca 60 30 150 46C 79 15 215 389
Hypothalamus 5 Ca 50 20 400 20 50 (50%) 293
Supraoptic 2 Ca 64 60 250 65 200 110
Supraoptic 2 Ca 60 30 400 42 50 (84%) 119
Paraventricular 10 Ca 59 33 402 67 12 100 (67%) 253
Midbrain and pons
Area postrema 5 Ca 65 10 36 72 10 (100%) 157
Cochlear nucleus 2 Ca 75 40 400 59M 89 500 (50%) 101
Cerebellum
Purkinje 10 Ca 60 20 400 40C 79 23 3,300 110 189
Dorsal root
ganglion
DRG 2.5 Ca 65 35 2,000 82 100 (95%) 436
DRG 10 Ba 60 25 41C 60 25 330 295
Nodose ganglion 5 Ca 60 20 400 48 30 400 54
Nodose 10 Ca 55 15 400 29C 44 30 226
Pelvic ganglion 10 Ca 50 25 500 38T 57 10 231
Olfactory 2.6 Ca 57 36 50 82 429
Olfactory 3 Ca 70 40 60 45C 66 100 (71%) 200, 201
Retina
Rod bipolar 10 Ca 60 30 35 36T 59 30 1,000 (80%) 316
Cone bipolar 10 Ca 60 20 66 35T 60 55 1,000 (85%) 316
Oligodendrocytes 20 Ba 50 25 150 63 10 47
The divalent cation concentration used to measure the currents is shown. Properties of the current-voltage relationship are described in terms
of the voltage threshold where currents become detectable, the voltage where they peak (IV peak), and the maximum amplitude observed (Imax).
Methods used to calculate midpoints of the activation curves (V0.5) are denoted with the following superscripts: C, chord conductance; T, tail
currents; M, I/Imax. The midpoint of the steady-state inactivation curve is reported (h). Kinetics of inactivation (inact) can vary with test potentials;
therefore, the values shown represent the voltage-independent rate, which was typically measured at 10 mV. WCurrents decayed in a biexponential
manner; to simplify comparisons, a weighted tau was calculated according to the following equation W A11 A22, where A represents the
fraction of current decaying with the respective time constant. The values shown for recovery from inactivation represent the voltage-independent
rate, which was typically measured at 90 mV. Tail current kinetics continuously vary with repolarization potential, so when possible the values
shown represent those obtained at 90 mV. In some cases recovery from inactivation was found to be biphasic, so the values represent the tau of
the fast component, the fraction of channels that recover fast (in parentheses), and the tau of the slow component. Nickel responses represent either
the IC50 value obtained or the concentration tested and the percent block observed (in parentheses). LGN, lateral geniculate nucleus; MDN,
magnocellular dorsal nucleus; LM, lacunosum-moleculare of hippocampus; DRG, dorsal root ganglion; inact., inactivation; recov, recovery.

value is considerably more negative than the apparent more selective than it is. In summary, block by 10 50 M
threshold of activation, indicating that channels can inac- Ni2 can be used to implicate Cav3.2 channels, but addi-
tivate at potentials where they do not appear to open. tional evidence, such as a slowly deactivating tail current,
Similar h values have been obtained with the recombi- is required to rule out Cav2.3 channels. Both these crite-
nant Cav3 channels (see Table 8), where this closed-state ria, plus PCR, were recently applied to show the expres-
inactivation has been studied in greater detail (127). Al- sion of Cav3.2 in sympathetic ganglion neurons (231).
though most HVA channels inactivate at more positive The temperature sensitivity of T-type currents has
potentials, R-type and recombinant Cav2.3 channels inacti- been measured in thalamic neurons (85), DRG neurons
vate over a similar voltage range as T-type channels (334). (305), N1E-115 neuroblastoma cells (298), and GH3 cells
The time course of recovery from inactivation is an (343). Heating from 22 to 37C caused over twofold in-
important property of T-type channels. LTS are often creases in current amplitudes and accelerated channel
triggered after an inhibitory postsynaptic potential activation and inactivation. In contrast, heating did not
(IPSP), and this is due to fast recovery of T-type channels affect the position of the I-V curve (305). The effects of

during the IPSP (deinactivation), followed by their open- temperature have been found to be nonlinear, making it
ing as the membrane returns to its resting potential. Re- difficult to calculate the effect of a 10C increment (Q10)
covery is often measured by varying the time between in temperature (298, 343). In the linear range, Q10 values
(interpulse) an inactivating pulse and a test pulse. In most with an average of 2.5 have been found for effects on
studies the interpulse voltage was 90 mV, and similar amplitude, activation kinetics, and inactivation kinetics.
results are obtained at more negative voltages. In con- The pH sensitivity of T-type currents has been stud-
trast, recovery is slower at potentials where channels can ied in CA1 hippocampal neurons (405), ventrobasal tha-
also inactivate. There is considerable variability in the lamic neurons (365), and cardiac myocytes (83, 414).
reported values for recovery, ranging from 100 to 3,300 ms Acidification of the external solution decreased current
(Table 2). Although most studies found that recovery amplitudes, whereas alkalinization had the opposite ef-
followed a monoexponential time course, others have fect. In contrast, T-type currents are relatively insensitive
found evidence for a biexponential process. This discrep- to changes in intracellular pH (405). The effect of external
ancy might be due to differences in the duration of the pH is in part mediated by changes in the single-channel
inactivating pulse, where long pulses allow channels to conductance, which when measured with 110 mM Ca2
accumulate in a second inactivated state from which they increased from 3.5 pS at pH 6 to 10.8 pS at pH 9 (414).
recover slowly. As observed in sensory neurons (53), Small changes in pH have also been shown to shift the
recombinant channels recover with a monoexponential voltage dependence of activation and inactivation; chang-
time course from short pulses and with a slower, biexpo- ing pH from 7.3 to 6.9 shifted both of these parameters by
nential time course from long pulses, although this prop- 3 mV, while increasing the pH to 7.7 had the opposite
erty is less pronounced for Cav3.3. Differences in recovery effect (365). Larger shifts in the voltage dependence of
kinetics of T-type currents between different neurons can activation (15 mV) have been observed using larger
also be due to which Cav3 isoform they express (209). Of shifts in pH (from 7.5 to 9.8) (83). The observed pKa
the three isoforms, Cav3.1 channels recover the fastest values were 7, indicating that T-type currents will be
(120 ms from short pulses), and this time course is affected by modest changes in extracellular pH (365, 414).
similar to that observed for recovery of both native T-type
currents (300 ms; Table 2) and LTS (150 ms; Table 1). 2. Slices
Early studies found that LVA channels were more
sensitive to block by 50 100 M nickel than HVA chan- LVA currents have been characterized in slices pre-
nels (124, 152, 298). Subsequent studies found that many pared from numerous brain regions (Table 3). The most
neuronal T-type channels required 10-fold higher con- striking difference between these data and that obtained
centrations for block, with many studies reporting IC50 with isolated neurons is that currents are much larger in
values of 300 M (Table 2). Studies on recombinant slices. This difference has been attributed to loss of chan-
channels provided an explanation for this diversity; only nels that were localized on dendrites (97). In fact, T-type
Cav3.2 channels are highly nickel sensitive (IC50 10 M), channels appear to be preferentially localized to dendrites
while Cav3.1 and Cav3.2 are 20-fold less sensitive (230). (77, 135, 195, 199, 328).
Recombinant HVA channels also differ in their Ni2 sen- T-type currents recorded from slices also appear to
sitivity, with Cav2.3 being relatively sensitive (455). Al- activate and inactivate at more negative potentials than
though the mechanisms of block of recombinant HVA and observed in isolated neurons. The average threshold in
LVA channels are complex and subject to multiple inter- slices was 70 mV, and peak currents were observed at
pretations, a consistent finding is that block is greatest at 45 mV (Table 3), while in isolated neurons threshold
potentials near threshold. This voltage dependence would was 60 and the peak occurred at 30 mV (Table 2). This
exaggerate block of LVA channels, making nickel appear difference has been attributed to poor voltage clamp of

TABLE 3. Properties of T-type currents in brain slices
IV
Divalent, Threshold, Peak, Imax, V0.5, h, inact, recov, Reference
Brain Region mM mV mV pA mV mV ms ms Nickel, M Nos.
Cerebellum 0.5 60 33 690 51M 86 14 100 (19%) 292

Thalamus
LGN 1 65 53 1,500 61M 77 26 100 200 (53%) 93
LGN 2 70 45 1,812 61M 84 17 151
Laterodorsal, IT,f 2 Ca 80 55 670 86 32 398
Laterodorsal, IT,s 2 Ca 80 65 630 98 55, 69 25 (50%) 398
Hypothalamus MDN 2 83 50 1,051 70M 82 12 100 500 (100%) 302
Brain stem respiratory 1.5 65 40 550 59M 81 200 (50%) 107
Dorsal horn 2 Ba 54 20 1,000 45M 86 14 200 (91%) 347
The voltage dependence of activation was estimated by normalizing the current observed at each test potential to the maximum ( M ) (I/Imax),

fit with a Boltzmann function, then reported as the midpoint voltage (V0.5). A single concentration of nickel was tested, and the percent block is
shown in parentheses. LGN, lateral geniculate nucleus; MDN, magnocellular dorsal nucleus.
neurons in slices (97). Consistent with this suggestion, the myocytes isolated from dog Purkinje (367), rabbit sino-
voltage dependence of single T-type channels recorded atrial node (152), cat atrium and latent pacemaker cells
from dendrites is similar to that observed in isolated (462), hamster ventricle (362), and rat atrium (446). In
neurons (256). general, the currents were small, displaying a peak cur-
In summary, T-type currents are found in neurons rent density below 3 pA/pF. In contrast, prominent T-
throughout the brain, with particularly large currents type currents (10 pA/pF) have been recorded from
found in thalamic, septal, and sensory neurons. Their myocytes isolated from shark (271), chick (202), finch
activation near the resting membrane potential and their (48), and mollusks (452).
fast recovery from inactivation allow them to generate Calcium channels were originally classified by their
LTS. Their preferential localization in dendrites suggests inactivation kinetics: rapidly inactivating or transient
T-type channels play an important role in synaptic inte- channels were called T type, while slowly inactivating, or
gration. long-lasting channels were called L type (303, 306). It
should be noted that this only holds for currents carried
C. Heart by Ba2, as cardiac L-type currents carried by Ca2 can
inactivate at a similar rate as T-type currents. This is due
Cardiac T-type currents have been the focus of many to calcium-induced inactivation of the L-type channel,
studies due to their possible role as a pacemaker current. which appears to be mediated by calmodulin tethered to
These studies have established that T-type current densi- the carboxy terminus of the channel (111). This inhibition
ties are highest in conduction and pacemaker cells, and is triggered with every heartbeat as Ca2 entry triggers a
much reduced or nonexistent in ventricular myocytes. larger release of Ca2 from internal stores (37). In con-
T-type currents are highest near birth and gradually de- trast, Ca2 currents through T-type channels inactivate a
cline but may reappear in pathological conditions. Cur- bit slower than Ba2 currents (116, 410), which excludes
rents are larger in lower species and have a wider distri- a similar regulation by calmodulin. Another major differ-
bution. For example T-type currents are prominent ence between these channels is that L-type channels con-
throughout the guinea pig and hamster heart but virtually duct Ba2 threefold better than Ca2, whereas T-type
absent in adult ventricular myocytes of rat, cat, and dog. channels conduct both equally well (discussed further in
Although Cav3.2 was cloned from a human heart cDNA sect. IIJ).
library, T-type currents have not been detected in human A proposed physiological role for cardiac T-type
myocytes (39, 235, 313). channels is as a pacemaker current during diastolic de-
Cardiac T-type currents were initially described in polarization. These studies have been hampered by the
dog atrium (32) and guinea pig ventricle (283, 303). These lack of a selective blocker, relying heavily on the ability of
studies showed that cardiac T-type currents resembled 40 M nickel to block T- but not L-type currents (152,
those recorded from neurons, but differed from L-type 298). Current-clamp studies of spontaneous action poten-
channels in terms of their kinetics (transient), pharmacol- tials showed that nickel slowed the late phase of depolar-
ogy (dihydropyridine insensitive), conductance of diva- ization, and hence slowed the firing of rabbit sinoatrial
lent cations (Ca2 Ba2), lack of regulation by -adren- nodal cells (SAN). Similar results have been obtained by a
ergic agonists, and smaller single-channel conductance of number of groups using rabbit SAN (98, 353) or cat latent
Ba2. Subsequent work has established their presence in pacemaker cells (462). Tetramethrin (0.1 M) was re-

ported to produce selective block as well, and to produce nantly express Cav3.2 channels. In contrast, in situ hybrid-
similar effects on pacemaker cycle (152); however, sub- ization of mouse heart suggests that Cav3.1 channels pre-
sequent studies failed to confirm this selectivity (165). dominate, although both isoforms were readily detected
Similarly, a number of studies have found lower sen- and both were enriched in SAN relative to atrium (49). It
sitivity and selectivity for nickel (Table 4). Two possible should be noted that distinct in situ probes might differ in
explanations for these disparate results are 1) that heart their ability to detect their cognate sequence, making
expresses two isoforms of the T-type channel, Cav3.1 and such comparisons difficult. PCR amplification of mouse
Cav3.2, and these isoforms differ in their sensitivity to heart detected the expression of a splice variant of Cav3.1
nickel, and 2) that isoform expression is age and species that differs in the III-IV loop (91).
dependent. Cloned Cav3.2 channels are blocked at 20-fold Expression of T-type currents in developing heart
lower concentrations than Cav3.1 (or Cav3.3), displaying has also been studied. T-type currents are readily detect-
an IC50 of 10 M (230). There is a strong correlation able in neonatal hearts, increase slightly to a peak be-
between nickel sensitivity and Cav3.2 expression, suggest- tween postnatal days 4 and 8, then decline slowly to a

ing that native Cav3.2 channels are as sensitive as the steady-state (236, 246). In other studies T-type currents
cloned channel (323). This suggests that nickel (100 were only detected in neonatal rats, disappearing by post-
M) can be used to implicate Cav3.2 channel expression. natal day 21 (236). In contrast to L type, the biophysical
Expression in atrium appears to be species specific: properties of T-type currents are unchanged during neo-
Cav3.1 appears to predominate in rat and mice (91, 236), natal development. Changes in nickel sensitivity have not
while in guinea pigs it is Cav3.2 (323). In fact, careful been investigated but may be informative. No difference
measurement of the nickel dose response in guinea pig in T-type current density was detected in SAN myocytes
atrium revealed a biphasic response, with 80% of the from newborn (310 days old) and adult (41 48 days old)
channels being blocked with an apparent IC50 of 23 M, rabbits (329). Expression of Cav3.2 mRNA is higher in
while 20% of the channels were inhibited with an IC50 of fetal human heart than adult, whereas Cav1.2 shows the
1,350 M. Rabbit and cat SAN cells appear to predomi- opposite pattern (331).
TABLE 4. Properties of T-type currents in peripheral tissues

Divalent, Threshold, IV Peak, Imax, V0.5, h , inact, deact, Reference
Tissue mM mV mV pA/pF pA mV mV ms ms recov, ms Nickel, M Nos.
Heart
Atrium 5 Ca 50 30 0.3 24 20 32
Atrium 5.4 Ca 50 30 1.0 75 40M 57 15 197 50 (66%) 444
Atrium 2 Ca 50 30 2.6 42C 68 13 1.3 160 236
Atrium 1.8 Ca 50 30 350 24T 59 23 323
Atrium 2.5 Ca 60 40 115 38C 59 10 116
SAN 2.5 Ca 47 10 2.1 88 23C 75 3.2 144 40 (100%) 152
Latent pacemaker 2.7 Ca 50 20 3.3 90 31C 57 40 (54%) 462
Purkinje 2 Ca 60 30 1.7 391 48C 68 10 100 100 (90%) 165
Purkinje 5 Ca 40 25 2.9 700 47M 37 4 83 (67%) 50 (47%) 410
444
Ventricle 5.4 Ca 50 10 100 11 100 (100%) 129
Ventricle 20 Ba 50 30 5.8 858 43C 77 13 362
Skeletal muscle 1.8 Ca 55 42C 63 13 45
10 Ca 45 15 3.6 400 24C 41 16 224 (65%) 5.4 38
5,160
Spermatocytes 10 Ca 60 20 6.5 218 47C 64 7 1.6 118 200 (75%) 350
Adrenal
Glomerulosa 20 Ba 45 30 7T 56 7 82
Fasciculata 10 Ca 50 15 8.2 272 17G 50 18 1.7 400 (50%) 20 287
4,800
Pituitary
Pars intermedia 10 Ca 50 15 150 5T 1.8 84
Lactotropes 5 Ca 48 12 90 28M 66 15 100 (80%) 240
Melanotropes 11 Ba 50 20 30 61 19 204
Gonadotropes 20 Ca 40 20 75 27C 61 15 3 262
Pancreas
-Cell 10 Ca 40 16T 21 2.5 320
-Cell 15 Ba 60 30 35 14M 100 (100%) 25
Definitions are as in Table 2. Methods used to calculate activation curves are denoted with the following superscripts: C, chord conductance;
T, tail currents; G, constant-field equation of Goldman-Hodgkin-Katz; M, I/Imax. In some cases recovery from inactivation was found to be biphasic,
so the values represent the tau of the fast component, the fraction of channels that recover fast (in parenthesis), and the tau of the slow component.
SAN, sinoatrial node.

Cardiac hypertrophy appears to trigger a return to that lower blood pressure (356). In contrast, T-type chan-
the neonatal pattern of gene expression (388), leading to nels may be localized near SR in atrial pacemaker cells of
reexpression of T-type channels in rat and cat ventricular the cat (180). These authors suggested a novel pacemak-
myocytes (173, 268, 308). RNase protection assays indi- ing mechanism where Ca2 influx through the T-type
cate that Cav3.1 transcripts are upregulated in a rat model channel triggers subsarcolemmal Ca2 sparks, which in
where hypertrophy is induced by infarction (173). Al- turn activate Na/Ca2 exchange currents that depolarize
though levels of Cav3.2 transcripts were not examined, the membrane to threshold.
the sensitivity of reexpressed currents to nickel block A role for T-type channels in triggering atrial natri-
suggests a contribution of Cav3.2 channels as well (173, uretic peptide (ANP) secretion has been inferred from the
268). T-type currents are also reexpressed in dedifferen- effects of mibefradil (236, 434). Evidence that mibefradil
tiated rat ventricular myocytes (114). T-type currents acted on T-type channels was provided by the observation
have been found to be increased twofold in cardiomyo- that L-type blockers were much less potent blockers, or
pathic Syrian hamsters relative to other hamster strains stimulated ANP secretion.

(46, 362). Studies using 8-mo-old animals found that the
voltage dependence of activation and inactivation was
shifted to more negative potentials in ventricular myo- D. Kidney
cytes from cardiomyopathic animals (362). In contrast,
studies using 1-day-old hamsters found that only inactiva- Relatively little is known about the physiological
tion was shifted (46). The relevance of these observations roles of T-type channels in kidney. T-type currents have
to human pathologies is unclear as T-type currents have only been recorded from smooth muscle cells (SMC)
not been detected in myocytes isolated from normal isolated from rat interlobular and arcuate arteries (143).
atrium (235) or diseased ventricle (39, 337). Heterogeneity in Ca2 currents was observed, with ap-
Calcium influx through voltage-gated channels acti- proximately one-third of the freshly isolated myocytes
vates intracellular Ca2 release channels, leading to larger displaying only T-type currents (T-rich), one-third only L
elevations in intracellular Ca2 and hence muscle con- type, and the rest a mixture. Currents in T-rich cells were
traction. The role of L-type channels in this process is well some of the largest ever recorded for cardiovascular
established. T-type channels are also capable of triggering smooth muscle (156 pA in 2.5 mM Ca2; Table 5), which
this CICR, albeit much more weakly in both cardiac (370, allowed their recording in physiological Ca2 solutions.
461) and skeletal muscle (133). T-type current-induced Consistent with their assignment as T-type channels, cur-
contraction developed more slowly, suggesting that these rents activated and inactivated at voltages 20 mV more
channels are not localized near SR stores as are L-type negative than observed for L-type currents. In contrast to
channels. This observation coupled with the lower ex- most T-type currents, these SMC currents activated (I-V
pression of T-type channels in working myocytes indi- peak 10 mV) and inactivated (h 50 mV) at voltages
cates that they do not play a major role in excitation- that were slightly more positive, which might be charac-
contraction coupling. Consistent with this notion, terized as mid-voltage activated. They also inactivated
mibefradil has little effect on cardiac inotropy at doses about twofold slower during a pulse (inact 46 ms at 10
TABLE 5. Properties of LVA currents in smooth muscle

Divalent, Threshold, IV Peak, h , inact, Reference
Tissue mM mV mV Imax, pA mV ms Nickel, M Nos.
Arteries
Coronary 10 Ba 60 10 125 65 14 130, 131
Coronary 20 Ba 40 30 250 55 332
Aortic 20 Ca 60 30 140 80 20 600 3
Aortic 1.5 Ca 60 30 100 65 339
Aortic 20 Ba 40 12 24 339
Mesenteric 10 Ba 40 30 63 372
Rabbit ear 110 Ba 30 10 20 55 34
Rat tail 20 Ba 50 38 46 428
Veins
Portal 5 Ca 45 10 263 50 13 246
Organs
Lung 10 Ca 50 10 200 448
Uterus 1.8 Ca 60 30 300 70 453
Bladder 1.8 Ca 56 10 200 60 100 (100%) 382
Colon 2 Ca 60 10 385 58 20 30 (50%) 445
Definitions are as in Table 2.

mV). Atypical currents with similar properties have been nifedipine affected glomerular filtration rate. Mibefradil
reported for SMC isolated from rat portal vein (315) and has also been reported to decrease renin secretion in rats
from the terminal branches of guinea pig mesenteric ar- with renal artery clips, but had no effect on renin secre-
teries (291). tion from isolated juxtaglomerular cells (423). In conclu-
Quite surprisingly, the human tissue that expresses sion, these studies suggest that T-type channels on affer-
the most mRNA for Cav3.2 is the kidney (90, 438). In ent and efferent glomerular arterioles may be an
contrast, both Cav3.1 and Cav3.3 are predominantly ex- important therapeutic target (89). A possible advantage to
pressed in brain (228, 288, 289, 324). Sktt and co-workers a T-selective antihypertensive drug is that it may also
(11, 156) have studied the distribution Cav3.1 and 3.2 in prevent glomerular damage (192).
kidney and concluded that Cav3.1 was in the tubules,
while Cav3.2 was in renal smooth muscle (11, 156). RT-
PCR indicated that mRNAs for Cav3.2, and to a lesser E. Smooth Muscle
extent Cav3.1, were expressed in rat (but not rabbit)

glomerular afferent and efferent vessels, and vasa recta. In addition to L-type currents, SMCs isolated from
The same study reported that K-induced contraction of arteries, veins, and organs have also been reported to
perfused rabbit afferent arterioles could be blocked by have LVA currents. With the exception of the kidney
low concentrations of mibefradil (IC50 10 nM) that results noted above, cardiovascular SMCs have tiny LVA
should be selective for T-type channels. Nickel could also currents. Therefore, most studies have had to use very
block this response; however, the concentrations re- high concentrations of charge carrier, which shifts gating
quired (1 mM) would be expected to block almost all Ca2 to positive potentials, and currents have only been char-
channels. Similar results were obtained with rat isolated acterized in terms of their I-V relations and sensitivity to
perfused hydronephrotic kidneys: either 1 M mibefradil holding potential. These characteristics are not sufficient
or 100 M nickel (a relatively selective dose) dilated to establish an LVA current as T type, since HVA currents
afferent and efferent arterioles constricted with ANG II actually represent a spectrum of mid- to high-voltage-
(314). Selectivity of mibefradil for T-type channels was activated currents. HVA currents also inactivate over a
demonstrated by coadministration with the L-type wide range of potentials, with some R-type currents inac-
blocker nifedipine. In these experiments 1 M nifedipine tivating over the same voltage as T-type channels. These
caused a modest dilation of the efferent arteriole but did concerns take on additional weight with the finding that
not prevent the more pronounced dilation induced by some vascular SMCs express non-L-type HVA currents
mibefradil. (291) such as P-type currents (155). Native P-type cur-
Cav3.1 was detected in dot blots of human fetal, but rents can be mid-voltage activated (33). In addition, a
not adult, kidney (288). RNase protection assays of rat nickel- and mibefradil-sensitive LVA current has been
kidney regions indicated that Cav3.1 was predominantly described in colonic myocytes that gates similar to T-type
expressed in the inner medulla (11). RT-PCR of microdis- channels but has different permeability properties (214).
sected nephron segments indicated that Cav3.1 was ex- LVA channels can be classified as T type by their slow tail
pressed in distal convoluted tubules, connecting tubules, currents, which surprisingly have not been reported, and
and collecting ducts. This study also examined the distri- by their single-channel properties, which have been re-
bution of Cav3.1 protein using a polyclonal antibody ported (34, 130, 450). Because T-type channels inactivate
raised against the first 22 amino acids of the deduced at the resting membrane potentials of most SMCs (75 to
Cav3.1 sequence (11). Immunoreactivity was detected in 60 mV, reviewed in Ref. 168), it has been hard to discern
the apical domains of the distal convoluted tubules and in their physiological role. Perhaps they contribute to basal
principal cells of connecting tubules and inner medullary intracellular Ca2 concentrations via their window cur-
collecting ducts. Preincubation of the antibody with pep- rent, or after transient hyperpolarizations induced by
tide blocked the signal; however, Western blots were not spontaneous transient outward currents (STOCs).
presented to demonstrate the selectivity of the antibody. LVA currents have been found in the following arte-
These results suggested that T-type channels, along with rial SMCs: coronary (130, 281, 332), aortic (3), mesenteric
endothelial calcium channels (ECaC), might be involved (150, 311, 372), rabbit ear (34), rat tail (327, 428), and
in Ca2 reabsorption. middle cerebral arterioles (167). RT-PCR detected the
Calcium channel blockers are widely used in the expression of both Cav3.1 and Cav3.2 in rat mesenteric
treatment of hypertension. It is generally accepted that arterioles (150). Single-channel studies have established
they work by blocking L-type channels in vascular smooth the presence of 7.5-pS T-type channels in freshly isolated
muscle, leading to vasodilation. Recent studies suggest guinea pig coronary SMCs (130). This study also reported
that part of their effect may be mediated by changes in that whole cell LVA currents could only be observed in
renal hemodynamics (172). In intact dogs, both nifedipine half of the cells. In contrast, LVA currents were never
and mibefradil increased renal blood flow, whereas only detected in freshly isolated coronary SMCs from rabbits

(269) or humans (332). LVA currents were found if the as separate from L-type channels (30, 81). Developmental
cells were cultured, and these currents appear to be T studies of mice and rats have shown that the T-type
type based on their voltage dependence and 1:1 conduc- current is only found in embryonic and newborn muscle
tance of Ca2 and Ba2 (332). Similar results have been and disappears by 3 wk of age (29, 38, 142). Concomi-
obtained with aortic SMCs; T-type currents are expressed tantly, the slow L-type current increases. Studies on the
in proliferating cultures, but neither in confluent cultures muscular dysgenesis (mdg) mouse clearly established
(3, 339) nor in freshly isolated cells (220). In fact, T-type that the T- and L-type channels were encoded on separate
currents were only detected in cells that were in G0 or G1 genes (30, 67, 212), and it is the L-type channel (Cav1.1)
phases (220), leading to the hypothesis that they might that plays a critical role in excitation-contraction coupling
play a role in proliferating SMCs (338). Consistent with (395). Recent studies have shown that the T-type current
this notion is the finding that mibefradil could reduce is important for myoblast fusion (45). In particular, it was
neointima formation following balloon injury to rat ca- shown that T-type channels could generate sufficient win-
rotid arteries (355). However, it should be noted that dow currents to alter intracellular Ca2 concentrations,

T-type currents were not observed in SMCs prepared from and trigger fusion. This hypothesis was further supported
injured rat aorta, although a transient downregulation of by pharmacological experiments using low concentra-
L-type channels was documented (333). tions of Ni2 and amiloride, and by antisense oligonucle-
LVA currents have also been detected in SMCs iso- otides directed against Cav3.2 (45). The selective expres-
lated from veins, such as saphenous (450), portal (246), sion of Cav3.2 in skeletal muscle fibers has also been
and azygous (282). T-type single-channel currents were demonstrated using single-cell PCR (38). The electrophys-
recorded in saphenous vein SMCs (450). Isradipine (PN iological properties of these currents (Table 4) and Ni2
200 110) was found to block T-type currents of rat portal sensitivity closely resemble recombinant Cav3.2 currents,
vein with an IC50 of 0.5 M, while L-type currents were although differences have been noted in kinetics of acti-
blocked at 1 nM (246). The sensitivity of these T-type vation, inactivation, and recovery from inactivation (38).
currents was greater than observed in other preparations Early studies also noted kinetic differences as a function
(see sect. VA), suggesting that Cav3 isoforms may differ in of development (142), suggesting that these differences
their pharmacology. Current-clamp recordings revealed are not due to rapid events such as phosphorylation, but
that portal vein SMCs could fire action potentials that rather slower events such as gene transcription of auxil-
resembled those observed with cardiac myocytes (246). iary subunits (see sect. IVE).
Isradipine (10 nM) inhibited the plateau phase with no
effect on the rising phase, suggesting that T-type channels
might play a pacemaker role in these cells. G. Sperm
LVA currents have been described in SMCs from
bronchi (185, 448), ileum (373), colon (445), bladder Calcium channels appear to play a role in mediating
(382), and uterus (453). The results with pregnant human the egg-induced acrosome reaction that precedes fusion
uterine SMCs are notable because the T-type currents (3 (for review, see Ref. 94). T-type channels have been im-
pA/pF in 1.8 mM Ca2) were larger than the L-type cur- plicated in this process, although other Ca2 conduc-
rents (453). These currents activated and inactivated tances may be involved (134, 435). One reason for this
(h 70 mV) at potentials typical of most T-type cur- uncertainty is that it is virtually impossible to patch clamp
rents (Table 5). Current-clamp recordings indicated that a mature sperm (335). Therefore, electrophysiological stud-
depolarizing pulse could trigger an action potential; how- ies have used spermatogenic cells, which are primary
ever, the threshold (20 mV) was higher than observed spermatocytes in the pachytene stage. The only voltage-
with neuronal LTS. Sizable (200 pA) T-type currents dependent Ca2 channels in these cells are T type, and
were also described for porcine bronchial smooth muscle, their biophysical (350) and pharmacological properties
being detected in 30% of the cells, but absent from tra- (15) have been well characterized. PCR results indicate
cheal SMCs (448). LVA currents (1.4 pA/pF) were found that Cav3.2 is the predominant isoform expressed in hu-
to disappear when bladder SMCs were cultured (382). man testis (182, 375). Consistent with this result is the
Concomitant with this loss was a shift in the threshold for high sensitivity (IC50 34 M) with which nickel blocks
action potential generation from 25 to 15 mV, sugges- the spermatocytic T-type current (15), although it should
tive of a role for T-type channels. be noted that Sertoli cells also express nickel-sensitive
T-type currents (225). Similar concentrations of nickel
also block the acrosome reaction (121, 375). Similarly, the
F. Skeletal Muscle following compounds have been found to block sper-
matocyte T-type channels and the acrosome reaction at
Newborn rodent skeletal muscle was also used in similar concentrations: isradipine (PN 200 110), nifedi-
early studies to establish the existence of T-type channels pine, pimozide, amiloride, mibefradil, W-7, and trifluoper-

azine (13, 15, 121, 247, 350, 375). Spermatocytic T-type erate spontaneous action potentials and Ca2 spikes, lead-
channels appear to be regulated by tyrosine and calmod- ing to secretion of hormone (see Ref. 406 and references
ulin-dependent protein kinases (14, 247). therein). Hormonal regulation of these LVA currents sup-
ports the idea that these currents are involved in secretion
(see sect. III). The voltage- and time-dependent properties of
H. Endocrine Tissues these currents are similar, but not identical to other native
T-type currents (Tables 2 and 4). Notably the peak of the I-V
1. Adrenal curve is slightly depolarized. In addition, one study reported
that the LVA current was fivefold larger in Ba2 than Ca2
T-type channels have been implicated in the secre-
(379), a property typically ascribed to HVA currents. As
tion of aldosterone (76) and cortisol (109). Calcium cur-
suggested previously, not all LVA currents are carried by
rents, and their regulation by secretagogues, have been
T-type channels, and other criteria must be applied (19, 33,
extensively studied in cells isolated from bovine adrenal
376). One such discriminating property is their tail deactiva-
zona glomerulosa and fasciculata. Glomerulosa cells ex-

tion kinetics, and slowly deactivating T-type currents are
press a mixture of T- and L-type channels (82), whereas
clearly present in pituitary cells (84). Similarly, these chan-
fasciculata cells predominantly express T-type channels
nels can be distinguished at the single-channel level by their
(22, 287). Although these cells can be considered nonex-
conductance for Ba2, and again, pituitary T-type currents
citable due to their lack of Na channels, they are still
have been clearly identified (203). In situ hybridization stud-
capable of generating Ca2-dependent action potentials
ies suggest all three Cav3 isoforms are expressed, although
(22). In contrast, adrenal chromaffin cells express HVA
Cav3.2 was the predominant isoform detected (393). Consis-
Ca2 and Na currents but no LVA currents (16).
tent with this result, T-type currents were reported to be
Serum K is regulated by secretion of aldosterone
nickel sensitive, with 80% of the current blocked by 100 M
from adrenal glomerulosa cells. Glomerulosa cells are
NiCl2 (240). Currents recorded from GH3 cells are not nickel
excellent K sensors and depolarize after small increases
sensitive (IC50 777 M), suggesting that this tumor-de-
in serum K, which in turn leads to the activation of
rived cell line most likely expresses Cav3.1 channels (160). In
T-type channels, Ca2 influx, and stimulation of aldoste-
conclusion, T-type channels appear to play a pacemaker role
rone synthesis and release. For example, increasing K
in anterior pituitary, although they may also be involved in
from 2 to 5 mM causes the resting membrane potential of
stimulus-secretion coupling (258).
isolated cells to depolarize from 97 to 78 mV (76).
Aldosterone secretion is also stimulated by ANG II, an
effect mediated by stimulation of T-type channel activity 3. Pancreas
(discussed in sect. IIIB).
T-type channels appear to play a similar pacemaker role
Cortisol secretion from adrenal zona fasciculata cells
in insulin secretion from pancreatic -cells of the islets of
is regulated by ACTH, which depolarizes these cells by
Langerhans. Increases in plasma glucose and its metabolism
inhibiting a K conductance (286). Depolarization would
in -cells leads to increases in cellular ATP and inhibition of
activate T-type channels, leading to a rise in intracellular
KATP channels (300). Closure of KATP channels initiates the
Ca2, and ultimately cortisol synthesis and secretion. Cor-
pacemaker cycle by depolarizing the -cell membrane to
tisol secretion can be inhibited by a variety of blockers
about 55 mV. Riding on top of this slow plateau depolar-
including mibefradil, and this block occurs over the same
ization are rapid calcium-dependent spikes (reviewed in Ref.
concentration range as their block of the T-type channels
352). Insulin secretion can be blocked by a wide variety of
(109, 141, 345). ACTH may also regulate the expression of
agents, indicating that these spikes activate L-, P-, and R-type
T-type channels (22).
currents (238, 258, 418).
Adrenal T-type channels are encoded by Cav3.2. In
T-type currents in pancreatic -cells have been char-
situ hybridization of rat and bovine glands detected high
acterized at the whole cell (Table 4; Refs. 25, 320) and
levels of Cav3.2 expression in the cortex, with only trace
single-channel level (17, 348). Expression is species de-
amounts of Cav3.1 and Cav3.3 (358). The biophysical prop-
pendent, with little or no expression in rodents, but
erties and sensitivity to Ni2 block of both glomerulosa
readily detectable in humans (25, 426). Therefore, it is
and fasciculata cells are also consistent with their being
notable that LVA currents were found in -cells from
encoded by Cav3.2 (287, 358).
diabetes-prone NOD mice (426). The expression of LVA
currents in mouse cells has been reported to be stimu-
2. Pituitary
lated by a 6-h treatment with cytokines (25 U/ml of inter-
LVA Ca2 currents have been recorded from cells of the leukin-1 plus 300 U/ml of interferon-; Ref. 427). How-
anterior and intermediate lobes of the pituitary gland includ- ever, the I-V curve for the cytokine-induced current
ing (84, 392, 439) lactotropes (240), corticotropes (261), peaked between 10 and 20, while under similar re-
gonadotropes (379), and thyrotropes (204). These cells gen- cording conditions this group found that bona fide, slowly

deactivating, T-type currents peaked between 20 and sively T-type currents, allowing characterization of these
10 mV (426). Human -cells express both LVA and HVA channels in the absence of contaminating HVA currents
currents, and in 20% of the cells tested, the LVA current (Table 6). The study of T-type currents in cell lines has
was dominant (25). Voltage-clamp recordings indicated advanced our understanding of gating, permeation, and
that 100 M Ni2 could block the LVA current, while pharmacology (73, 160, 368). For example, Chen and Hess
current-clamp recordings indicated that it slowed action (73) developed models of gating using recordings from
potential frequency. Similar studies using the rat insuli- 3T3 fibroblasts. They also showed that the T-type currents
noma cell line INS-1 found that low concentrations of of NG108 15 cells had different kinetics, leading them to
nickel could completely block T-type currents (IC50 30 predict the existence of multiple channel isoforms. A
M) and partially block insulin secretion (42). Nickel- similar conclusion was reached from studies on TT cells,
sensitive T-type currents (IC50 3 M) have also been which also recovered from inactivation much more
recorded from the mouse insulinoma cell line NIT-1 (426). slowly than observed previously (44). Differences in the
Li and co-workers (463) also cloned a splice variant of pharmacology of T-type channels also suggested the ex-

Cav3.1 from an INS-1 cDNA library. The high sensitivity to istence of distinct channel isoforms (160). The cloning of
nickel block suggests that Cav3.2 channels are also ex- multiple Cav3 isoforms that differ in their kinetics, phar-
pressed, and this conclusion is supported by Northern macology, and recovery from inactivation supports this
analysis (438). hypothesis (209, 228, 404).
I. Cell Lines J. Single-Channel Recordings
T-type currents have been reported in a number of Single-channel studies provided conclusive evidence
cell lines such as 3T3 fibroblasts (73) and many lines of that T-type channels were distinct from HVA Ca2 chan-
tumor origin, such as neoplastic B lymphocytes (128), the nels (63, 303, 306). T-type channels were distinguished by
related mouse neuroblastoma-derived lines N1E-115 (298, their smaller currents, insensitivity to dihydropyridines,
368), NG108 15 (196, 334), 140 3 (144), N18 (397), and and voltage dependence of activation and inactivation. In
ND723 (213); human neuroblastoma lines IMR-32 (65) addition to providing criteria for resolving T-type cur-
and SK-N-MC (18); rat pituitary lines GH3 (160, 270); rents, single-channel studies have provided insights into
human TT cells (also called h-MTC) and rat 6 23 (clone 6) both hormonal regulation of activity and gating.
cells from medullary thyroid tumors (43, 44); human Y79 With isotonic Ba2 as the charge carrier (110 mM),
retinoblastoma cells (24); AT-1 from mouse atrium (351); the single-channel current at a test potential of 0 mV is
rat smooth muscle lines A7r5 and A10 (272); and the 0.3 pA for T-type channels (Table 7) and 1.5 pA for
pancreatic insulinoma lines INS-1 (rat; Ref. 42), HIT-T15 L-type channels. Single-channel conductance is defined as
(hamster; Ref. 259), and NIT-1 (mouse; Ref. 426). Many of the slope of the line relating single-channel amplitudes
these cell lines were reported to express almost exclu- versus test potential and is commonly reported in pico-
TABLE 6. Properties of T-type currents in cell lines

Divalent, Threshold, IV Peak, Imax, V0.5, h, inact, deact, Reference
Cell Line mM mV mV pA mV mV ms ms recov, ms Nickel, M Nos.
Human
TT 10 Ca 40 15 211 27T 51 16 2 1,900 5 (57%) 44
IMR-32 10 Ca 50 20 80 15 200 (52%) 65
Y79 20 Ba 50 20 87 32C 40 20 24
Rat
C-cell 6-23 10 Ca 50 15 500 31M 57 20 2 1,260 5 (22%) 43
GH3 10 Ca 50 20 600 33G 71 20 5 200 (80%) 777 160
1,000
Mouse
MAb-7B 2.5 Ca 65 35 80 82 16 128
N1E-115 50 Ba 50 20 300 47 298
NG108-15 10 Ba 60 15 140 27C 53 20 2 334
ND7-23 10 Ba 50 30 119 67 18, 32 845 100 (60%) 213
3T3 20 Ba 50 20 500 65 10 2 100 73
AT-1 2.5 60 35 500 48 C
64 14 2.5 150 (73%) 351
1,200
Definitions are as in Table 2. Methods used to calculate activation curves are denoted with the following superscripts: C, chord conductance;
T, tail currents; G, constant-field equation of Goldman-Hodgkin-Katz; M, I/Imax.

seimens (pS). When recorded in Ba2, the conductance of they approach from the intracellular side of the channel
T-type channels is 7 8 pS (Table 7), which is smaller than (255, 363). The selectivity sequence for monovalent
that observed for either N-type (1315 pS) or brain L-type cations through T-type channels is Li Na K
channels (2528 pS) (118, 125, 417). Somewhat surpris- Rb Cs (128, 255). In fact, T-type channels begin to
ingly, all three families have a similar Ca2 conductance. pass outward K currents at physiological concentrations
For example, the L-type channel conductance drops of Ca2 and voltage (28 mV; Ref. 128). HVA channels
threefold in Ca2 (149, 367). Recombinant Cav2.1 and can also pass outward currents, but this requires depolar-
Cav2.2 channels also preferentially conduct Ba2 (2- ization of the membrane beyond 70 mV (161). Our un-
fold), whereas Cav2.3 conducts both ions equally well derstanding of Ca2 channel permeation is far from com-
(56). In contrast, native T-type channels conduct Ca2 and plete, and subject to multiple interpretations (275, 442).
Ba2 (and Sr2) equally well (64, 104, 367, 368). The Single-channel currents have also been studied at
conductance plots differ in their relative position, with Ca2 concentrations closer to the physiological levels,
T-type channels gating at more negative ranges, and ex- displaying a single-channel conductance of 2 pS in 5 mM

trapolating to a reversal potential of approximately 40 (64) and 4.6 pS in 10 mM Ca2 (21). Whole cell studies
mV, while HVA currents appear to reverse at 60 mV (34, using varying concentrations of Ca2 indicate that cur-
52, 118, 124, 194, 203, 213, 367, 417). rents increase sharply over the 110 mM range, then
Estimates of permeability based on reversal poten- saturate at higher concentrations. These studies have al-
tials yield the opposite rank order, with Ca2 being more lowed estimates of the affinity of the open channel for
permeable than Ba2 (128). Similar results have been Ca2. The average KD value from seven studies is 4.9 mM,
obtained using recombinant T-type (363) and native L- with considerable variability (range 0.310 mM) (4, 22,
type channels (161) and suggest that Ca2 binds with high 54, 152, 160, 389, 429).
affinity in the pore and permeates when displaced by a The transitions between open and closed states have
second Ca2. A very interesting property of all voltage- been studied in detail in DRG neurons (64), cardiac myo-
gated Ca2 channels is that they become highly perme- cytes (104), fibroblasts (73), and N1E-115 neuroblastoma
able to Na in the absence of Ca2. Apparently Na are cells (368). T-type channels respond to depolarizing
not capable of displacing the bound Ca2, and hence do pulses by opening in bursts, that is, channels open and
not permeate. This block is unidirectional, since monova- close many times, become silent for a while, and at some
lent cations can displace Ca2 bound in the pore when voltages may burst again. The fast opening and closings
TABLE 7. Single-channel properties

Current at 0 Conductance, Reference
Source Divalent, mM mV, pA pS Selectivity Nos.
Rat DRG 40 Ca 0.2 5.2 Ca Ba 64

Chick DRG 110 Ba 0.4 8 124
Mouse DRG 60 Ba 0.25 7.2 Ba Sr 217
Hippocampal CA3 pyramidal 95 Ba 0.3 7.8 118
Septum and diagonal band 100 Ba 0.2 7.8 147
Cerebellar Purkinje 110 Ba 0.3 9.0 52
Pituitary menalotropes 110 Ba 0.4 8.1 203
Hypoglossal motoneuron 110 Ba 0.3 7 417
Retinal ganglion 96 Ba 0.36 8 194
Cardiac ventricle 110 Ca 0.33 6.8 Ba Ca 104
Cardiac Purkinje 110 Ba 0.4 9.0 Ba Ca 367
Ear artery SMC 110 Ba 0.3 6.9 34
Coronary artery SMC 110 Ba 0.38 7.5 130
Saphenous vein 90 Ba 0.35 8 450
Adrenal glomerulosa 110 Ba 0.25 8.7 Ba Ca 27
Pancreatic -cells 100 Ba 0.3 6.4 348
Neuroblastoma N1E-115 60 Ba 0.25 7.2 Ca Ba Sr 368
Neuroblastoma ND7-23 110 Ba 0.25 7.9 213
Neuroblastoma 140-3 110 Ba 0.3 6.84 144
Low divalent
DRG 5 Ca 2 64
Cardiac ventricle 10 Ca 0.16 4.7 21
Coronary artery SMC 10 Ca 0.16 4.7 21
Coronary artery SMC 10 Ba 0.2 4.6 130
Single-channel properties were measured from cell-attached patches using the indicated concentration of divalent cation as charge carrier. The
slope conductance of high-voltage-activated channels is similar to T-type channels when recorded in Ca2 solutions but can be distinguished by their
current amplitudes at a test potential of 0 mV. DRG, dorsal root ganglion; SMC, smooth muscle cell.

can be modeled as transitions between a closed state C the whole cell observation that steady-state inactivation
(C4 in Fig. 3) and an open state O. The transition rates out can be observed at potentials more negative than channel
of the open state determine the mean open time, which is opening. This inactivation is slow and voltage dependent
typically 0.52 ms (Table 7). Multiple bursts are seen (364). Inactivation from open (or the proximal closed
during depolarizing pulses just above the threshold for state C4) has similar kinetics and limits the channel to one
channel opening. At more depolarized potentials a single burst per sweep. Whole cell recordings suggest that this
burst is observed, indicating that channels entered an rate is relatively voltage independent at potentials above
inactivated state. Analysis of the time a channel spends in 20 mV. Several models of T-type channel gating have
closed states reveals a bimodal distribution; closings been proposed that can simulate the observed activity.
within a burst are short (1 ms), whereas the time be- One reason these models differ is that they are based on
tween bursts is longer (10 ms). Channels can also open disparate results. Unresolved questions include the fol-
partially, leading to a subconductance state that has a lowing: Does the voltage dependence of the mean open
smaller current (27, 64, 104). In contrast to other ion time explain the slowly deactivating tail currents ob-

channels (66), the gating behavior of this subconductance served at the whole cell level (64)? One study found that
state was found to be similar to the main conductance open times were shorter at more negative test potentials
state (64). (64), another found no difference (104), while two found
A distinguishing kinetic feature of T-type channels is that they increased (217, 368). To complicate matters
that whole cell current traces recorded during an I-V more, two studies have found evidence for a second type
protocol produce a criss-crossing pattern, where succes- of opening of longer duration (27, 147). Discrepancy in
sive traces cross each other. This is largely due to slow mean open time measurements has been attributed to the
activation kinetics at threshold potentials (60 to 40 low signal-to-noise ratio of channel openings (73). A re-
mV). At the single-channel level, T-type channels show a lated question is: Does the burst duration vary with test
long latency to the first opening, especially at threshold potential? Most studies report the burst duration at a
potentials, where 40 ms may pass before the first opening.
single voltage, although one study found that it increased
This delay presumably reflects slow transitions between
from 5 ms at 40 mV to 14 ms at 10 mV (104). These
closed states and can lead to a sigmoidal rise in the whole
models might also differ because of T-type channel het-
cell current. This rate is voltage dependent, such that
erogeneity, as evidenced by the distinct gating behavior of
channels open faster at more depolarized potentials (104).
the three recombinant Cav3 channels (228). Recently, a
In most sweeps (70%) the channel does not open at
model has been proposed that can simulate the gating
all, resulting in a blank sweep (73, 104). This suggests that
behavior of both recombinant Cav3.1 and Cav3.3 channels,
channels might inactivate without opening. Such closed
although different rate constants for the transitions are
state inactivation has been observed in other channels
and is particularly prominent in Cav2 channels (321). required (127, 364). The model assumes that depolariza-
Closed state inactivation also provides an explanation for tion leads to sequential movement of each of the four S4
voltage sensors (see sect. IVB), culminating in a final
closed state that precedes the open state. The open state
can either deactivate (return to C4) or inactivate (IO).
Closed state inactivation is allosterically coupled to S4
movement and becomes prominent after three S4 sensors
have moved (Fig. 3). Movement of the fourth voltage
sensor does not affect the inactivation rate, so open chan-
nels inactivate at the same rate as closed channels in C3
and C4 states. The speed of voltage sensor movement and
subsequent transitions among closed states appears to be
slower in Cav3.3 than Cav3.1, leading to slower activation
FIG. 3. Simplified gating scheme. The model represents transitions kinetics. The channels also differ in their equilibrium
of T-type channels between rested (R), closed (C), inactive (I), and open between C4 and O states, with Cav3.3 favoring C4 while
states (O). Changes in the membrane potential are thought to induce
movement of the S4 voltage sensors, which leads to rearrangements in
Cav3.1 favors O. Therefore, Cav3.1 channels inactivate
the channel structure. Movement of three of the four S4 regions is more from open states, whereas Cav3.3 channels inacti-
sufficient to induce closed states that inactivate relatively quickly. The vate more from closed states. The model accounts for
proximal closed state (C4) can also show closed state inactivation or
lead to channel opening. Single T-type channels show a propensity to most, but not all, of the observed properties of T-type
open in bursts during a prolonged depolarization, and this represents channels; in particular, it does not attempt to explain
transitions between C4 and O states. The model has been simplified from multiple open states or ultraslow (1 s) inactivation
the version presented by Frazier et al. (127) by omission of C1, C2, I1, and
I2. The effects of voltage suggest that S4 sensors must move back toward (127). In summary, voltage-gated Ca2 channels can be
their original position before channels can recover from inactivation. distinguished by their single-channel properties, with T-

type channels showing a smaller conductance in Ba2 and anterior pituitary lactotrophs by 25 40% (239, 240). Do-
equal conductance of Ca2 and Ba2. pamine shifted the h curve from 63 to 77 mV (5 Ca)
and accelerated current decay during the pulse. It had no
effect on voltage dependence of activation. Dopamines
III. REGULATION
effects were reversible, mimicked by the dopamine ago-
Among the first criteria used to distinguish LVA from nist (D2 class) bromocryptine (10 nM), and prevented by
HVA channels was their resistance to rundown in solu- the D2 antagonist sulpiride (100 nM). Inhibitory regulation
tions that lack cAMP, ATP, and Mg2, suggesting that LVA could be abolished by omitting GTP from the intracellular
channels were not phosphorylated by cAMP-dependent solution or by pretreatment with pertussis toxin. Inhibi-
protein kinase (115). Consistent with this hypothesis, hor- tory modulation could also be disrupted by inclusion of
mones that stimulate adenylyl cyclase, such as the -ad- antibodies that specifically recognize the -subunit of Go,
renergic agonist isoproterenol, had no effect on T-type while antibodies against Gi subunits had no effect (239).
currents recorded from either canine atrial myocytes (32), Parallel studies showed that D2 receptors coupled to po-

rabbit sinoatrial nodal cells (152), canine Purkinje cells tassium channels via a different G protein, Gi-3.
(166, 410), guinea pig ventricular myocytes (415), rabbit Dopamine inhibited rat adrenal glomerulosa T-type
ear artery (35), or guinea pig hippocampal CA3 neurons current by 33% (103). Inhibition was mediated by the D1
(117). Stimulation of L-type currents via cAMP-dependent receptor class as evidenced by 1) inhibition with the D1
pathways provided a positive control for most of these agonist SKF 82958; 2) block by the D1 antagonist SCH
studies. Although some studies have reported a small 23390; 3) but not by the D2 antagonist spiperone. Dopa-
stimulation of T-type currents by isoproterenol (9, 283), mine-mediated inhibition was blocked by agents that dis-
this may be due to either incomplete separation of T- from rupt signaling by 1) G proteins [2 mM guanosine 5-O-(2-
L-type currents (as evidenced by BAY K 8644 stimulation) thiodiphosphate) (GDPS)], 2) adenylyl cyclase (100 M
or due to secondary regulation induced by changes in 2,3-dideoxyadenosine), and 3) protein kinase activity
internal Ca2 concentrations (411). In contrast to the (10 M H-89). Consistent with an action through D1 re-
apparent lack of protein kinase A (PKA) regulation, hor- ceptors, dopamine caused a stimulation of cAMP forma-
monal regulation of T-type currents has been reported in tion. Somewhat surprisingly, bath application of 8-bromo-
many systems. Both inhibition and stimulation have been cAMP had no effect on basal T-type currents but could
reported, and in some cases the same agonist did both. block the effects of dopamine. Addition of 100 nM G
For example, the GABAB agonist baclofen has been re- subunits to the pipette solution did not affect T-type
ported to cause stimulation at 2 M but inhibition at 100 currents. However, G inhibition was observed after
M (361). addition of 1 mM 8-bromo-cAMP to the bath. These re-
sults suggested that G subunits might bind directly to
A. Hormonal Inhibition the T-type channel.
Somatostatin (10 nM) was found to inhibit T-type
Many hormones and neurotransmitters have been currents by 50% in rat somatotrophs (72). Somatostatin
reported to inhibit T-type currents, including dopamine, caused the currents to decay faster during a sustained
serotonin, somatostatin, opioids, ANP, and ANG II. Al- pulse and shifted the midpoint of the steady-state inacti-
though most of these studies were performed on isolated vation curve (h) curve from 52 to 63 mV (charge
neurons, regulation has also been noted in secretory cells carrier was 5 mM Ca2). This regulation was abolished by
of the pituitary and adrenal. pertussis toxin. Neurotensin (200 nM) and substance P
Dopamine inhibition of T-type currents has been de- (200 nM) inhibited by 25% the T-type currents from rat
scribed in DRG neurons (260), adrenal glomerulosa cells cholinergic neurons (263). The -opioid agonist Tyr-D-Ala-
(312), pituitary melanotrophs (204), lactotrophs (240), Gly-Met-Phe-Gly-ol (DAGO) was found to inhibit DRG
and cells from the pars intermedia (309). Chick DRG currents by 50% (360). Enkephalin was found to inhibit
currents were shown to be inhibited 60% by either 10 M T-type currents 20 40% in the neuroblastoma cell line
dopamine or norepinephrine (260). Inhibition occurred NG108 15 (196). In these cells there was no effect of
with no apparent effect on kinetics. Recovery upon wash- either the phorbol ester 4-phorbol 12-myristate 13-ace-
out was not observed in whole cell recordings. Similar tate (PMA), arachidonic acid (10 M), or forskolin. Bra-
inhibition was observed at the single-channel level, al- dykinin (0.1 M) inhibited 57% of the T-type current from
though in these experiments washout was observed. Sin- ND723 cells, a cell line derived from DRG (213). This
gle-channel current amplitude was not affected, suggest- study also showed that ()-baclofen (1 M) could inhibit
ing that inhibition was due to a reduction in the currents by 56%, whereas guanosine 5-O-(3-thiotriphos-
probability of opening (Po). phate) (GTPS) inhibited by 20%. Pertussis toxin treat-
Dopamine (10 nM) inhibited T-type currents of rat ment did not block baclofen inhibition. Baclofen (20 M)

has also been reported to inhibit the T-type currents in due to an increase in the probability of opening of each
hippocampal neurons by 53% (126). channel, as there was no change in the single-channel
Muscarinic inhibition of T-type currents from hen conductance, which was 7.1 pS in a solution containing 95
granulosa cells has also been reported (425). In contrast mM Ba2. Under similar conditions, carbachol inhibited
to most studies that report 40% inhibition, this study single L-type currents. Carbachol (50 M) and serotonin
reported 90% inhibition. Part of this difference may be due (30 M) were also shown to stimulate hippocampal T-
to their use of the perforated patch technique, which is type currents 54 and 61%, respectively (126). These effects
expected to preserve the integrity of the intracellular were blocked by the appropriate receptor antagonists and
milieu. The effect of carbachol was blocked by atropine. were quickly reversible. Serotonin appeared to shift the
In contrast, muscarinic agonists have been reported to I-V to the left (peak shifted from 25 to 30 mV), but this
have no effect on LVA channels in cholinergic neurons (5). effect was not quantitated (126). Erythropoietin increased
ANP (3 nM) inhibited T-type currents of bovine ad- T-type currents 20% in the human neuroblastoma cell line
renal glomerulosa cells by 28% (274). Inhibition was de- SK-N-MC (18). It also stimulated increases in intracellular

pendent on the holding potential, with little block when Ca2 that was dependent on external Ca2 but not inter-
membrane potential (VM) was 90 mV, but near complete nal stores. Norepinephrine acting via -adrenoceptors
block when VM was 45 mV. This voltage dependence of was reported to increase T-type currents in canine Pur-
block also caused a 10 mV shift of the h curve to more kinje cells by 69% (410).
negative potentials. In contrast, ANP did not affect the Endothelin has been reported to stimulate T-type
voltage dependence of activation as measured using tail currents from both cardiac and smooth muscle myocytes
currents (26). The effects of ANP were also studied at the (129, 181). In neonatal rat ventricular myocytes, endothe-
single-channel level. Single T-type channels were identi- lin-1 displayed an EC50 of 1.3 nM and a maximal stimula-
fied by their 8.3-pS conductance. Inhibition was accom- tion of 54% (129). Heat inactivation of the endothelin, or
panied by a decrease in the number of active sweeps, with omission of GTP from the pipette, occluded the effects.
no change in the amplitude of single openings. The ability The stimulation induced by 10 nM endothelin was
of bath-applied ANP to modulate channels recorded in the blocked by 0.2 M staurosporine or 20 M H-7. Protein
cell-attached mode indicated that ANP induced the pro- kinase C (PKC) agonists stimulated the currents directly,
duction of a soluble second messenger. and this could be blocked by H-7 as well. In guinea pig
ANG II and the AT2 selective ligand CGP-42112 inhib- smooth muscle myocytes, endothelin stimulated the ac-
ited by 25% the T-type current in NG108 15 cells (58, 59). tivity of a 12-pS channel, that may or may not be carried
Inhibition was associated with a 5-mV shift in the I-V but through T-type channels (181). Endothelin has been re-
no effect on the h curve. Inclusion of 3 mM GDPS in the ported to have no effect on T-type currents recorded from
pipette blocked the ANG II effect. In contrast, 3 mM smooth muscle cells from rat renal resistance arteries
GTPS did not block the ANG II inhibition. This result is (143).
surprising since this concentration of GTPS should have Acetylcholine stimulated T-type currents in NIH 3T3
maximally stimulated G protein signaling. Washout was cells that had been transfected with recombinant m3 and
not reported. A role for tyrosine phosphorylation in the m5 muscarinic receptors (322). Stimulation was modest
signaling pathway was suggested by the ability of 50 M (25%) and was accompanied by a leftward shift in the I-V
sodium orthovanadate to disrupt ANG II modulation of relation. Parallel assays demonstrated that cAMP levels
the T-type current. were increased. T-type currents were also stimulated by
treatments designed to stimulate cAMP-mediated signal-
ing, such as 500 M 8-bromo-cAMP or 10 M forskolin.
B. Hormonal Stimulation Likewise, the PKA inhibitor 10 M Rp-adenosine 3,5-
cyclic monophosphothionate (RpcAMPS) inhibited basal
T-type currents can also be stimulated acutely by a activity and occluded the response to acetylcholine.
variety of hormones. One of the first studies showed that These results suggested that T-type channels might be
10 M serotonin could stimulate rat spinal motoneuron directly phosphorylated by PKA. An apparent contradic-
T-type currents by 66% in a slice preparation (36). Also in tion to this hypothesis was that T-type currents were not
a slice preparation, dorsal horn neurons were found to be regulated in cells transfected with the m1 muscarinic
stimulated by substance P (1 M), although some neurons receptor, although these cells showed robust increases in
were inhibited (347). Cholinergic agonists also stimulate cAMP. The authors speculated that m1 receptors might
T-type currents, and this was demonstrated at the single- simultaneously activate inhibitory PKC pathways that ob-
channel level. Carbachol and muscarine doubled the num- scured the PKA-mediated stimulation. First they showed
ber of openings of single T-type channels in adult guinea that the PKC activator phorbol 12,13-dibutyrate (PDBu;
pig hippocampal CA3 neurons (117). This stimulation was 0.5 M) directly inhibited T-type currents as observed in
blocked by 0.1 M atropine. The modulation was likely other cells types. Next they showed that preincubation

with the PKC inhibitor calphostin C (0.5 M) allowed up to 87% (361). Inhibition was accompanied by an accel-
m1-mediated stimulation of the T-type current. Basal T- eration of inactivation, such that the decreased from 22
type currents were not regulated by acetylcholine in cells to 18 ms. Neither cholera toxin pretreatment nor photore-
transfected with either m2 or m4. However, inhibition of lease of caged GDPS had any effect. Pertussis toxin
T-type currents, as well as inhibition of cAMP formation, pretreatment blocked GTPS inhibition, but not stimula-
could be observed in these cells after stimulation of ad- tion. GTPS has also been shown to inhibit T-type cur-
enylyl cyclase by 10 M forskolin. These results indicate rents (40%) of rat nodose ganglion neurons (148). This
that hormonal regulation of T-type channels may depend effect was blocked by pretreatment with pertussis toxin.
on both basal phosphorylation levels as well as cross-talk Somewhat surprisingly, G proteins do not appear to
between PKA and PKC pathways. mediate the regulation of T-type currents by serotonin
Barrett and colleagues (82) have extensively studied and nociceptin. Serotonin inhibition (25%) of T-type cur-
the stimulation of adrenal glomerulosa T-type Ca2 chan- rents has been observed in Xenopus sensory neurons
nels by ANG II. Initial studies established the presence of (383, 384). Inhibition was not observed when the T-type

T-type channels in these cells through the use of tail channels were recorded in the cell-attached patch config-
current analysis (82). Slowly deactivating currents gated uration, indicating that its effects were membrane delim-
with the voltage dependence expected of T-type channels, ited. Inhibitory regulation was not blocked by pertussis
while fast-deactivating currents behaved like HVA, L-type toxin pretreatment or inclusion in the pipette of GDPS,
channels. ANG II increased the T-type tail currents by 50% GTPS, or 5-guanylyl-imidodiphosphate (GMP-PNP). The
(82). The effects of ANG II were also studied at the ability of these manipulations to block the regulation of
single-channel level (273). ANG II increased both the HVA currents in the same neurons provided a convenient
number of active sweeps and increased the number of positive control. It was concluded that G proteins are not
openings per sweep, with no appreciable change in the involved in the serotonin inhibition of T-type channels. A
amplitude of single openings. The ability of bath-applied similar conclusion was reached in a study on the nocicep-
ANG II to modulate channels recorded in the cell-attached tin inhibition of DRG T-type currents (1). Nociceptin in-
mode indicated that ANG II induced the production of a hibited currents with an IC50 of 0.1 M, and in 22 of 77
soluble second messenger. The effects of ANG II were neurons it blocked 100% of the current at 1 M. Inclusion
blocked by the ANG II receptor antagonist saralasin (1 in the pipette of GTPS, GDPS, or AlF3 did not alter
M). ANG II also shifted the voltage dependence of acti- response, leading the authors to conclude that a G protein
vation by 10 mV to more negative potentials, with no is not involved. It should be noted that nociceptin had no
change in the h curve. Pretreatment of the cells with effect on T-type currents from small nociceptive trigemi-
pertussis toxin abolished these effects, indicating that nal ganglion neurons (51). The endogenous cannabinoid
ANG II receptors couple through G proteins, possibly Gi anandamide also blocks T-type currents independently of
(251). The final step in this signal transduction pathway G proteins and receptors (71).
appears to be phosphorylation of the T-type channel by The ability of pertussis toxin treatment to block reg-
calmodulin-dependent protein kinase II (CaMKII; see be- ulation indicates that Gi/Go proteins are involved. Support
low) (27, 252). However, not all studies have found an for this hypothesis comes from studies that used specific
ANG II stimulation of adrenal glomerulosa currents, with antibodies in the patch pipette to block regulation (239,
reports of either no effect (250) or frank inhibition (103, 251). Although the -subunits of G proteins have been
344). suggested to interact directly with T-type channels (103),
more studies are required to establish this point.
C. Guanine Nucleotides
D. Protein Kinases
As noted above, T-type currents are regulated by G
protein-coupled receptors. Therefore, this regulation With the notable exception provided by studies on
should require the presence of GTP and should be modi- fibroblasts (322), T-type currents appear not to be regu-
fied by activators (GTPS) and inhibitors (GDPS) of G lated by PKA. In contrast, T-type currents appear to be
proteins. Accordingly, no hormonal regulation was ob- inhibited by members of the PKC family and stimulated by
served when GTP was omitted from the patch pipette tyrosine kinases and CaMKII.
(129, 239) or when it was replaced with GDPS (59, 103). The effects of PKC have been evaluated by activating
The effects of GTPS are difficult to interpret be- the kinase directly with compounds such as diacylglyc-
cause it can activate all G proteins. With the use of erol 1-oleolyl-2-acetylglycerol (OAG), or various phorbol
photoactivation of caged GTPS in DRG neurons, low esters: PMA, 12-O-tetradecanoylphorbol-13-acetate (TPA),
concentrations were observed to stimulate T-type cur- or PDBu. Although many studies have found no effect of
rents by 54%, while higher concentrations inhibited them these compounds (310), both stimulation and inhibition of

activity have been reported. Studies on rat DRG neurons also blocked regulation. As observed with native chan-
found that 10 nM PMA inhibited the T-type current by nels, stimulation was accompanied by an 11-mV hyperpo-
27%. Likewise, the inactive analog 4-phorbol had no larizing shift in the voltage dependence of activation, with
effect. Of note, the PMA effect was not observed at room no shift in the h curve. In contrast, the activity of human
temperature, requiring preincubation at 29C or higher Cav3.1 was not regulated under similar conditions. These
(359). Similarly, 100 nM TPA was found to inhibit the studies strongly suggest that the modulatory phosphory-
T-type current by 26% in ventricular myocytes and Pur- lation site(s) reside directly on the 1-subunit of T-type
kinje cells. A phorbol ester analog that does not activate channels and that these sites are subtype specific, in this
PKC had no effect, while the protein kinase inhibitor H-8 case only found on Cav3.2. Although CaMKII regulation of
prevented TPA inhibition (411). In contrast, 50 M H-7 T-type currents has not been reported in other systems, it
was not able to block the inhibition of hippocampal T- might have mediated the 30% stimulation observed in
type currents by 10 M TPA or 5 M OAG (407). In cardiac myocytes (411).
addition, the TPA and OAG effects occurred faster than

would be expected (200 ms), leading Toselli and Lux
(407) to conclude that the compounds were blocking the E. Voltage
channel directly. PKC may in part mediate the inhibitory
effects of arachidonic acid (459). An intriguing property of many HVA Ca2 currents is
PKC-mediated stimulation of T-type currents has also that their activity can be increased, or facilitated, by
been reported (129). Currents in rat neonatal ventricular strong depolarizing pulses. These prepulses can induce
myocytes were stimulated 30% by either 0.2 M PDBu or channel conformations that are 1) more susceptible to
PMA. The inactive analog 4-PDBu had no effect. Stimu- phosphorylation, 2) have a lower affinity for divalent
lation was blocked by inclusion of 20 M H-7 in the cations in the pore, or 3) have a lower affinity for G
pipette. protein -subunits. The prepulse can also directly induce
The ability of CaMKII to stimulate adrenal T-type high activity gating modes (mode 2) of cardiac L-type
currents has been studied at both the whole cell and channels. Both native (below) and recombinant (140)
single-channel level, and recently with recombinant chan- T-type currents have also been reported to be facilitated
nels. Inclusion of Ca-CaM in the pipette shifted the volt- by prepulses.
age dependence of activation to more negative potentials The T-type currents of guinea pig coronary smooth
(252). The CaMKII inhibitor KN-62 or the synthetic pep- muscle myocytes were stimulated twofold by 200-ms pre-
tide inhibitor that corresponds to residues 290 309 pulses to voltages above 30 mV (131). Studies where the
blocked this effect. Stimulation was also demonstrated at prepulse potential was varied indicated that saturation
the single-channel level in both cell-attached and excised occurs at 10 mV. Use of longer prepulses (10 s) shifted
patches (27). In cell-attached patches, manipulation of this voltage dependency to the left, such that saturation
intracellular Ca2 led to an increase in channel activity occurred at 60 mV. Changing the duration of the pre-
that could be blocked by KN-62, but not the inactive pulse showed that the peak effect occurred between 160
analog KN-04. Channel activity could also be stimulated and 320 ms, which then decayed and was gone by 5 s. This
when excised inside-out patches were exposed to cal- potentiation was greater when the interpulse voltage was
modulin and appropriate concentrations of Ca2. This 100 compared with 80 mV, and at 36 versus 24C.
stimulation could be blocked with the autocamtide 2-re- Potentiated currents inactivated faster (inact 5.6 vs.
lated inhibitory peptide (AIP). Stimulation could also be 14.5 ms).
mimicked by the direct addition of a constitutively active Facilitation has also been observed in bone marrow
mutant of CaMKII. Heat-inactivated kinase had no effect. cells, where T-type currents were stimulated twofold by a
As observed previously with ANG II (273), this regulation 750-ms prepulse to 150 mV (330). Compared with the
appeared to be due to an increase in active channels. results obtained in smooth muscle, stronger depolariza-
These studies clearly establish that native bovine glo- tions were required; stimulation was not observed with
merulosa T-type channels are regulated by CaMKII. Adre- prepulses lower than 30 mV, and saturated at 100 mV.
nal cortical T-type channels are encoded by 1H, or Cav3.2 Changing the duration of prepulse (10-mV pulse)
(358). Recent studies have shown that calcium/calmodu- showed that half-maximal stimulation occurred at 250
lin can stimulate the activity of cloned human Cav3.2 ms, and saturated by 1,000 ms. Changing the interval
channels when coexpressed with CaMKIIc in HEK-293 between the prepulse and the test pulse showed that
cells (443). Stimulation could be blocked by CaMKII in- potentiation peaked after 1 s and decayed in a biexponen-
hibitors added to either the bath (3 M KN-62) or to the tial manner: 9.4 and 43.5 s. Potentiation was not affected
intracellular pipette used for whole cell recording (AIP, 2 by omission of ATP or GTP or bath application of non-
M). Replacement of pipette ATP with the nonhydrolyz- specific protein kinase inhibitors (e.g., 100 M H-7). Po-
able analog 5-adenylyl-imidodiphosphate (AMP-PNP) tentiated currents decayed slightly faster (inact decreased

from 17 to 13 ms; currents isolated by subtraction). These many groups, these approaches were not successful in
results suggest that voltage directly affects channel activ- isolating cDNAs encoding T-type channels.
ity without the involvement of second messenger signals. Progress in the sequencing of human, yeast, and C.
The opposite conclusion was reached in studies of bull- elegans genomes provided a novel library that could be
frog atrial cells (8). Amphibian channels are stimulated by screened with a computer, or in silico (Fig. 4B). Screening
-adrenergic agonists, and a prepulse enhances this stim- with the highly conserved P loop, or K channel signature
ulation. These effects were enhanced by GTPS and sequence, allowed Ketchum et al. (205) to identify a novel
ATPS, but blocked by either GDPS or pertussis toxin K channel sequence from Saccharomyces cerevisiae. We
treatment. From these studies it was concluded that G decided to try a similar approach using S5 segments. This
proteins are involved and that phosphorylation of G pro- led to the identification of two Ca2 channel-like proteins
teins may play a role as well. that were predicted from the C. elegans genome (325).
Facilitation of T-type currents from mouse sper- These proteins were homologous to the skeletal muscle
matogenic cells has also been reported (14). In this case, Ca2 channel, and this similarity was noted in the Gen-

prepulses could only stimulate activity 50% above control. Bank entry. We reasoned that there might be other se-
Prepulses to voltages greater than 30 mV were required quences in the GenBank that were similarly labeled and
to observe stimulation, and saturated around 60 mV. used a text-based search (Fig. 4B) to find sequences that
Short prepulses (10 ms) were capable of stimulating were similar to a calcium channel. This led to the iden-
currents, and this effect wore off very slowly. Membrane- tification of the human EST clone H06096 (324). Although
permeable inhibitors of tyrosine kinases (tyrphostins A47 this clone is 1.4 kb long, only a part of the 5-sequence was
and A25) stimulated basal T-type currents but had no in the database. This fragment had a very low sequence
effect on facilitated currents. This suggests that tyrosine identity to the S1 segment of other Ca2 channels, pre-
kinases may tonically inhibit activity in this system and cluding its direct identification by homology search. The
that a prepulse somehow reverses their activity. Consis- full sequence of H06096 showed that it might encode a
tent with this hypothesis, inhibitors of tyrosine kinase
protein with many of the hallmarks of voltage-gated chan-
phosphatases inhibited basal T-type currents and pre-
nels, notably the S4 voltage sensor sequence was well
vented voltage-induced potentiation.
conserved, and the pore loop sequence was highly homol-
In summary, T-type channels can be both stimulated
ogous to other Ca2 channels. Screening of the GenBank
and inhibited by hormones and neurotransmitters that are
with the H06096 sequence and the program BLAST iden-
coupled to the Gq or Gi/Go families. Regulation appears to
tified C54d2.5 as a C. elegans homolog. Similar to in vitro
be mediated by phosphorylation, with inhibition mediated
cloning where one walks through a cloning project us-
by PKC and stimulation by CaMKII. Additional studies are
ing probes to the ends, we screened the GenBank with the
required to establish the location of the modulatory phos-
phorylation site(s), whether G protein subunits are capa- 3-end of C54d2. This led to the identification of the EST
ble of interacting directly with the channel, and the mech- clone H19230. In vitro screening of a rat brain cDNA
anisms by which voltage regulates channel activity. library with H06096 allowed the cloning of Cav3.1 (324),
whereas use of this clone to screen a human heart library
led to the cloning of Cav3.2 (90). Screening of a rat brain
IV. MOLECULAR CLONING library with H19230 under low-stringency conditions al-
OF T-TYPE CHANNELS lowed us to identify not only rat versions of Cav3.1 and
Cav3.2, but also Cav3.3 (228). A similar approach was used
A. Cloning by Monteil et al. (288) to identify C54d2.5 and the H06096
and H19230 ESTs, which then allowed them to clone the
Cloning of high voltage-gated Ca2 channels began full-length human Cav3.1. Similar PCR approaches were
with purification and microsequencing of the skeletal also used to clone a human Cav3.2 cDNA (438) and mouse
muscle dihydropyridine receptor (396). Low-stringency and rat versions of Cav3.1 (211, 463). PCR approaches
screening of cDNA libraries with probes derived from the based on the C. elegans sequence C27f2.5 led to the
1-subunit of skeletal muscle (1S, or Cav1.1) led to the cloning of a distantly related channel gene whose func-
discovery of cardiac (1C, Cav1.2) and brain (1D, Cav1.3) tion remains unknown (227).
isoforms of the L-type family (Cav1; reviewed in Ref. 326). Sequencing of the human genome also played an
The more distantly related members of the Cav2 family important role in the cloning of T-type channels. The
were cloned during the brain era (Fig. 4A) using even Cav3.3 gene is located on chromosome 22, which was the
lower stringency hybridization (374). PCR provided an first chromosome to be completely sequenced (105).
alternative approach, and primers based on highly con- BLAST search with rat Cav3.1 identified the coding re-
served regions (IIIS5 and IVS5) were used successfully to gions of Cav3.3, which then allowed the design of PCR
isolate a fragment of Cav2.3 (357). Despite the efforts of primers to either clone its cDNA (228, 289) or to examine

2
FIG. 4. A timeline of Ca channel cloning. A: graph
illustrates the time of the first published reference to the
cloning of each of the 25 subunits. Skeletal muscle Ca2
channel subunits were purified, microsequenced, then
cloned using degenerate oligonucleotide probes based on
the protein sequence. This initial period has been called

the Muscle Era. Low-stringency screening of brain cDNA
libraries with the skeletal muscle probes led to the cloning
of other 1- and -subunits during the Brain Era. In the
final epoch, or In Silico Era, subunits were first identified
by database searching programs (BLAST) of Human and
Caenorhabditis elegans genome sequencing projects, then
cloned by standard in vitro methods. (Adapted from Ran-
dall A and Benham C. Recent advances in the molecular
understanding of voltage-gated Ca2 channels. Mol Cell
Neurosci 14: 255272, 1999.) B: flow diagrams describing
two ways of cloning in silico.
the splicing patterns of its gene (285). Proper identifica- exon 16; and 2) the III-IV linker, where three (or actually
tion of the 5- and 3-ends requires traditional cDNA clon- four, Ref. 229) variants are produced by a combination of
ing or specially designed PCR protocols. Recent studies insertion/deletion of exon 26 and use of an alternate
indicate that the human Cav3.3 mRNA contains an addi- 5-splice site in exon 25 (288). These variants differ in
tional exon that encodes 214 additional amino acids (140) their electrophysiological properties (68) and may ac-
than previously recognized (289). Studies comparing the count for the differences noted in the gating of T-type
full-length to truncated Cav3.3 channels revealed differ- channels between lateral geniculate (LGN) relay neurons
ences in channel expression and their ability to be mod- and interneurons (318). Splice variants of Cav3.3 have
ulated by a strong prepulse (140). been detected by PCR that differ in the I-II loop and
Genetic mutations in various Ca2 channel subunit carboxy terminus (285). Similarly, PCR has identified
genes have been associated with ataxic and epileptic splice variation in the III-IV linker of Cav3.2 in both rat
phenotypes (187). As a first step in exploring such a link, (231) and human (182).
we mapped the human and mouse genes for Cav3.1, Cav3 cDNAs have been cloned from rat, mouse, and
CACNA1G (324), and Cav3.2, CACNA1H (90). The genes human sources (Table 8). Although all three have been
were mapped using the Cambridge 4 radiation hybrid cloned from rat and human, cloning from mouse is still
panel and fluorescent in situ hybridization. The locations ongoing. Comparisons of their nucleotide sequences re-
of the human genes are shown in Table 8. Mutations in veal a high level of conservation between species. Al-
human Cav3 genes have yet to be described. though species differences in the predicted amino acid
All three T-type channel genes are alternatively sequence have been noted, some of these changes can be
spliced, producing variants that differ in their intracellular ascribed to either frameshifts in the reading, cloning ar-
loops (68, 284, 285, 463). Splicing produces Cav3.1 vari- tifacts, or differences in splicing. For example, it is likely
ants that differ in at least two locations: 1) the II-III loop, that a cloning artifact caused the deletion of three amino
where two variants are produced by insertion/deletion of acids from IIIS4 in a rat cDNA of Cav3.3 (279).

TABLE 8. Comparison of the three cloned T-type channels

Reference
Cav3.1 Cav3.2 Cav3.3 Nos.
Alias 1G, CavT.1 1H, CavT.2 1l

Gene name CACNA1G CACNA1H CACNA1l 249
Chromosomal location 17q22 16p13.3 22q12.313.2 90, 228, 324
Database entries*
Human NP_061496, O43497 AAC67239, O95180 XP_001125, Q9UNE6
Rat AAC67372, O54898 AAG35187, Q9EQ60 NP_064469, Q970Y8
Mouse NP_033913, Q9WUB8 NP_67390
Tissue expression Brain, ovary, placenta, heart Kidney, liver, adrenal cortex, Brain 90, 228, 288,
brain, heart 289, 324
Electrophysiology
Threshold, mV 70 70 70 209
Peak of IV, mV 30 30 30 209

Activation kinetics, ms 1 2 7 209
Inactivation kinetics, ms 11 16 69 209
Deactivation kinetics, ms 3.0 2.2 1.1 209
h, mV 72 72 72 209
Recovery from inactivation, ms 117 395 352 209
Conductance, pS; Ba 7.5 9 11 228, 324, 438
Pharmacology
Nickel (IC50, M) 250 12 216 230
Mibefradil (IC50, M) in 10 Ba2 1.2 1.1 1.5 267
Mibefradil (IC50, M) in 2 Ca2 0.27 0.14 267
* Accession numbers are for a representative GenBank entry followed by the SWISS-PROT entry. Results for conductance were obtained
in 110 mM Ba2, while all other electrophysiological measurements were made in 1.25 mM Ca2. Activation and inactivation kinetics were measured
with exponential fits to the currents elicited by test potentials to 10 mV. Deactivation kinetics were measured at 90 mV. Pharmacology was
measured in 10 mM BaCl2, or as noted, in 2 mM CaCl2 solutions.
B. Structure ward when the plasma membrane is depolarized. Little is

known about how this movement is transduced into chan-
Conservation of amino acid sequence (Figs. 1 and 5) nel opening. The pore loops are also well conserved,
and predicted secondary structure (Fig. 6) indicates that especially within the members of a particular ion channel
T-type channels are evolutionarily related to the -subclass. In voltage-gated Ca2 channels, the pore loops all
units of K, Na, and HVA Ca2 channels (184). Although contain the following signature sequence: T-x-E/D-x-W. In
little is known about the structure of Na and Ca2 HVA channels, all four repeats have a glutamate residue in
channels, recent advances with K channels provide a the pore, leading to the abbreviation EEEE. In repeats III
framework to interpret their structures. The most signif- and IV of Cav3 channels, the glutamate has been replaced
icant advance was the determination of the KcsA K by an aspartic acid residue, or EEDD. The importance of
channel structure by X-ray crystallography (102). This these residues in Ca2 permeation has been extensively
channel contains two transmembrane segments sepa- established in HVA channels (449), and recently with
rated by a pore loop (Fig. 6). The second transmembrane Cav3.1 (391).
segment forms the barrel walls of the pore, while the Site-directed mutagenesis studies on the S6 region of
selectivity filter is formed by the pore loops that dip Cav3.1 suggest that these segments play a similar struc-
partially into the barrel. In addition to this fundamental tural role in these channels as in KcsA (266). This pro-
motif, voltage-gated channels also contain four additional vides a theoretical basis for the use of the KcsA crystal
transmembrane segments, with one (S4) acting as a volt- structure to guide studies of drug binding pockets, which
age sensor. In K channels, four proteins assemble to in the case of Cav1.2 appear to be located within the
form one channel, while in Na and Ca2 channels a channel walls on the cytoplasmic side of the pore loops
single protein contains four of these modules, or repeats. (174).
Each transmembrane segment can be identified by a num- As shown in Figure 6, most of the channel is thought
ber to indicate the repeat it is in (traditionally a Roman to be intracellular. The extracellular loops are predicted
numeral) followed by its position within a repeat (Fig. 6). to be quite short, with the notable exception of the loop
For example, the third transmembrane segment of the connecting IS5 to the pore loop (98 103 amino acids).
third repeat can be abbreviated IIIS3. Among voltage- This extracellular loop contains six cysteine residues that
gated ion channels, the most highly conserved sequence are conserved in all three Cav3 channels and a number of
belongs to the S4 segments, where every third residue is possible sites for glycosylation. Neuraminidase treatment
positively charged. This region is thought to move out- of cardiac myocytes stimulates T-type (but not L-type)

currents threefold, suggesting that glycosylation of the rebral cortex, hippocampus, reticular nucleus, lateral ha-
channel inhibits its activity (116, 451). Despite the fact benula, and cerebellum. DRG neurons express both
that the sequences of intracellular loops differ consider- Cav3.2 and Cav3.3 mRNA (393). One study found that this
ably across Na and Ca2 channels, they have a similar expression was restricted to small and medium-sized neu-
overall size: long loops connect repeats 12 and 23 rons (393), while a second study reported that Cav3.3
(range 207375 amino acids for Cav3 channels), while transcripts were equally abundant in large DRG neurons
repeats 3 4 are connected by short loops (55 62 amino (454).
acids). Similarly, the amino termini are usually short
(100 amino acids), whereas the carboxy termini are
quite long (433 493 amino acids). The sequence of these D. Electrophysiology of Recombinant Channels
loops is poorly conserved, with the exception of se-
quences close to the end of the S6 segments (Fig. 5). In Expression of recombinant Cav3 channels leads to
HVA channels these loops are important for binding aux- the appearance of robust currents in a variety of heterol-

iliary subunits such as the -subunit, binding to regulatory ogous expression systems. Currents mediated by the
proteins such as calmodulin or the G protein -complex, cloned 1-subunit are nearly identical to those recorded
and are sites for protein phosphorylation (424). Although from native channels: they both open and inactivate at
the roles of these loops in T-type channels are presently potentials near the typical resting membrane potential,
unknown, it is likely they contain important regulatory recover rapidly from inactivation, and close slowly pro-
sites as well. ducing prominent tail currents (Fig. 7).
The currents mediated by rat and human isoforms of
the three Cav3 channels have been compared under a
C. Distribution variety of experimental conditions (209, 218, 228, 279). In
general these results agree quite well with each other,
The expression patterns of these genes in peripheral with the differences most likely due to recording condi-
tissues and brain have been studied using Northern blots, tions or sequence variability. For example, Cav3.1 and
RNA dot blots, and in situ hybridization (Table 8). Cav3.1 Cav3.2 currents inactivate (1.7-fold) faster with Ba2
mRNA is primarily expressed in human brain, but also in than Ca2 (209, 211). Similarly, recording conditions such
ovary, placenta, and heart (288, 324). A wider distribution as choice of charge carrier or length of depolarizing
was noted in dot blots prepared from human fetal tissues, pulses can affect estimates of both deactivation kinetics
with strong expression noted in kidney and lung (288). (211, 216, 430) and recovery from inactivation (430).
Cav3.2 mRNA is primarily expressed in kidney and liver, Therefore, comparisons under one set of conditions are
but also in heart, brain, pancreas, placenta, lung, skeletal the most relevant; Table 8 presents the results obtained in
muscle, and adrenal cortex (90, 438). Cav3.3 mRNA is 1.25 mM Ca2 (209), whereas Figure 7 results were ob-
almost exclusively expressed in brain (228); the only pe- tained in 5 mM Ca2.
ripheral tissues that showed expression on dot blots were All three Cav3 clones form LVA channels. Depolariza-
adrenal (but see Ref. 358) and thyroid (289). tion of the membrane to 70 mV is sufficient to trigger
The distribution of all three Cav3 channel transcripts channel opening, and the I-V curves for all three channels
in rat brain has been studied in exquisite detail using in peak around 30 mV (Fig. 7B). Some studies have found
situ hybridization (393), and similar patterns of expres- that Cav3.3 currents activate at slightly more positive
sion have been obtained using Northern blots of human potentials than either Cav3.1 or Cav3.2 (127, 289). Part of
brain mRNA (288, 289, 438). Their patterns of expression this difference can be ascribed to the methods used to
are complementary in many regions, with most brain estimate the voltage dependence of activation. Of note,
regions expressing more than one isoform. In fact, some not all the recombinant channels within a given prepara-
neurons may express all three genes as suggested by the tion gate at the same low potentials, with 40% of the
labeling of olfactory granule cells and hippocampal pyra- channels requiring much stronger depolarizations (10 to
midal neurons. Many brain regions showed heavy expres- 50 mV) for opening (288, 438). Activation curves con-
sion of Cav3.1 mRNA, including thalamic relay nuclei, structed using tail currents that detect both channels
olfactory bulb, amygdala, cerebral cortex, hippocampus, show a lower slope and higher midpoint of activation than
hypothalamus, cerebellum, and brain stem. Two separate other methods.
studies have confirmed this pattern of mRNA expression The kinetics of the currents are very voltage depen-
and extended the findings by showing a similar distribu- dent between 70 and 20 mV, producing a typical criss-
tion of Cav3.1 protein (88, 197). Cav3.2 mRNA expression crossing pattern of traces (Fig. 7A; Ref. 334). Therefore, it
was detected in olfactory bulb, striatum, cerebral cortex, is useful to compare the kinetics of the three channels at
hippocampus, and reticular thalamic nucleus. Cav3.3 10 mV or above (Table 8). Cav3.1 and Cav3.2 both acti-
mRNA expression is high in olfactory bulb, striatum, ce- vate relatively quickly at this potential ( 12 ms) and

FIG. 5. Alignment of the predicted amino acid sequence of human Cav3.1, 3.2, and 3.3 channels. Residues conserved
in all three sequences are shown in the consensus sequence (CON). The approximate location of the membrane-spanning
regions is indicated above the sequence. -Subunits bind to the I-II loop of HVA channels through a sequence termed the
-interaction domain (AID). The consensus sequence of the AID is shown above the homologous region in the Cav3
channels. The residues are colored according to the structural properties of the individual amino acids: red, positively
charged; green, negatively charged; blue, polar; yellow, nonpolar.

FIG. 5Continued
inactivate 10-fold slower ( 1116 ms). In stark con- are made using Xenopus oocytes (228), and it remains a
trast, Cav3.3 currents activate and inactivate very much mystery why Cav3.3 channels behave so differently in
slower. This difference is even greater when comparisons amphibian eggs than in mammalian cells.

FIG. 6. Structural models. A: hydrop-

athy plots of the KcsA and Shaker K
channels and repeat III of Cav3.1. The
hydropathy index varies from 3 to 1.
Letters above the plots indicate the rela-
tive position of the putative transmem-
brane segments (S1S6) and pore loops
(P). B: transmembrane segments are also
typically displayed in two dimensions as

snake diagrams, wherein the protein is
shown to snake its way in and out of the
membrane. C: a model of the three-di-
mensional structure of four-repeat ion
channels. The repeats are assumed to be
oriented in a clockwise manner based on
work with Na channels. The S5-P-S6
segments are thought to form a similar
structure as the S1-P-S2 segments of
KcsA, with the S6 segment forming the
walls of the channel. The S4 segment is
highly charged, so it is likely to be sur-
rounded by the other segments with wa-
ter filling the cracks. The S1S3 segments
are shown to form the exterior of the
channel. The transmembrane segments
are most likely composed of -helices,
but we have no clue to their orientation.
The intra- and extracellular loops are not
shown for clarity. D: a snake diagram
where each amino acid is represented by
a circle. Residues that are conserved in
all three Cav3 channels are shown with a
solid circle. (Adapted from Perez-Reyes
E. Three for T: molecular analysis of the
low voltage-activated calcium channel
family. Cell Mol Life Sci 56: 660 669,
1999.)
Recombinant Cav3 channels, like their native coun- ably similar. The midpoint of the h curves are 72 mV
terparts, close slowly producing prominent tail currents. (Table 8). Native T-type currents inactivate over a similar
The voltage protocol used to measure tail currents in- range. These studies indicate that T-type channels, as
cludes a short pulse to open channels, followed by repo- observed with other channels, can reach inactivated
larization to a voltage where they close (Fig. 7C). Al- states without passing through open states (364). In fact,
though all three Cav3 channels show slow tail currents during a depolarizing pulse up to 30% of Cav3.3, channels
upon repolarization to 90 mV, Cav3.1 are the slowest inactivate without opening (127).
( 3 ms), while Cav3.3 are the fastest ( 1 ms). In Cav3 channels recover rapidly from short-term inac-
contrast, HVA channels close 10-fold faster. tivation. Cav3.1 channels recover fastest, displaying a
Despite large differences in the rate at which they monoexponential recovery with a time constant of 100
inactivate, the voltage dependence of steady-state inacti- ms (Fig. 7, Table 8). In contrast, Cav3.3 channels recover
vation of the three recombinant Cav3 channels is remark- threefold more slowly, while Cav3.2 channels recover


FIG. 7. Electrophysiological properties of recombinant Cav3 channels. Recombinant channels were expressed in
human embryonic kidney cells (293) and recorded with the ruptured patch method using 5 mM Ca2 as the charge
carrier. A: the current-voltage (I-V) relationship is typically measured using a series of step depolarizations where the test
potential (80 to 10 mV) is increased 10 mV between runs. After each run the cell is held at 100 mV for at least 10 s
to allow recovery. Recordings were made using a stably transfected cell line expressing Cav3.2. B: I-V curve is a plot of
the current as a function of test potential. In this example the currents for each cell were normalized to the peak current
observed, and then averaged. Typical I-V relations are shown for human Cav3.1 (), 3.2 (), and 3.3 (E; same symbols for
all panels). The data were fit with an equation: G Gmax (Vm Vrev)/[1 exp(V1/2 Vm)/k], where G is conductance,
Vm is the test potential, Vrev is the apparent reversal potential, V1/2 is the midpoint of activation, and k is the slope factor.
C: tail currents are measured with a two-step protocol where the first step is designed to open channels (e.g., 2 ms to
60 mV), and the second step measures how fast they close at different repolarization potentials. Traces were obtained
with Cav3.1 and Cav3.3 after repolarization to 90 mV. Deactivation kinetics are measured by fitting tail currents with
exponential functions. D: deactivation kinetics vary with the repolarization potential. E: recovery from inactivation can
be measured using a three-step protocol where step 1 is a long depolarizing pulse used to inactivate channels, step 2 is
a repolarizing step to voltages where channels recover, and step 3 is a test pulse to measure the fraction of channels that
recovered. The duration of step 2 is increased in subsequent runs. Recovery is defined as the ratio of the peak current
in step 3 divided by the current in step 1. F: recovery time courses of the three human Cav3 channels. Data were fit with
an exponential to calculate the rate of recovery. Cav3.1 channels recover fastest with a of 100 ms, Cav3.3 are
intermediate (250 300 ms), while Cav3.2 recover slowest (400 ms). G: steady-state inactivation curves of the three
human Cav3 channels estimated with 15-s prepulses. Smooth curves represent fits to the data with a Boltzmann equation.
The foot of the activation curve is also shown. There is a small voltage range where these two curves overlap, leading
to the prediction that channels can produce steady-state (window) currents. H: fraction of channels open in the
steady-state can be estimated by multiplying the Boltzmann functions that describe activation and inactivation.
even more slowly (209). The channels also differ in their operationally defined as the overlap region between the
recovery from steady-state inactivation, and again, Cav3.2 activation and steady-state inactivation curves (Fig. 7, G
was found to recover the slowest (209). These results and H). All three Cav3 channels are predicted to generate
suggest the existence of multiple inactivated states, as window currents supported by 1% of the channels. Re-
noted previously for T-type currents from sensory neu- cording conditions and methods of analysis have a dra-
rons (53). These results also suggest that recovery kinet- matic effect on estimates of the window current, so these
ics can be used to differentiate currents carried by native estimates must be interpreted with caution. For example,
Cav3.1 from Cav3.2 (351). Fast recovery from inactivation analysis of Cav3.3 results obtained in Xenopus oocytes
is a critical property of native T-type channels that allows indicates that up to 2% of the channels can be open in the
them to participate in rebound burst depolarizations. steady-state. Evidence for window currents have also
The ability of T-type channels to open at similar been obtained in thalamacortical neurons, where they
potentials at which they inactivate suggests that they play an important role in signal amplification (441). Win-
might generate current under steady-state conditions. dow currents also appear to be important in determining
This is commonly referred to as a window current and is intracellular Ca2 concentrations (18, 69, 264). Expres-

sion of recombinant Cav3.1 or Cav3.2 was found to in- Studies using mock action potentials suggest that
crease basal Ca2 in HEK-293 cells, and this increase was most of the Ca2 influx through T-type channels occurs
blocked by appropriate concentrations of either mibe- after the peak voltage and corresponds to the tail current
fradil or nickel. Similarly, basal Ca2 is increased after (218, 276, 334). This Ca2 influx might play a role in spike
differentiation of the human prostatic cancer cell line repolarization and afterhyperpolarization mediated by
LNCaP, and this correlates with the expression of Cav3.2 apamin-sensitive Ca2-dependent K channels (440). Due
mRNA and currents (264). Hormonal regulation of the to differences in their rates of activation and inactivation,
T-type window current provides cells with another mech- the three T-type channels respond quite differently to
anism to regulate intracellular Ca2. In adrenal glomeru- trains of mock action potentials (218). Both Cav3.1 and
losa cells, ANG II increases T-type window currents by Cav3.2 channels inactivate rapidly during trains delivered
selectively shifting activation to more negative voltages at 20 Hz. In contrast, Cav3.3 continues to gate during
(273, 443), an effect mediated by CaMKII phosphorylation 100-Hz train frequencies. These differences suggest that
(discussed in sect. IIID). the three channels play distinct roles in determining neu-

Another distinguishing property of T-type channels is ronal firing patterns and signal integration. Cav3.1 and
that they have a lower Ba2 conductance than HVA chan- Cav3.2 channels are likely involved in short burst firing as
nels. Therefore, studies of cloned T-type channels have seen in thalamacortical neurons, whereas Cav3.3 channels
focused on the amplitude of single-channel currents in are likely involved in sustained burst firing as seen in
isotonic barium solutions. All three recombinant channels thalamic reticular neurons (70).
were found to have small single-channel currents, corre-
sponding to slope conductances in the 711 pS range
(Table 8). In contrast, Cav1.2 channels display slope con- E. Auxiliary Subunits?
ductances of 20 30 pS, while Cav2 channels are in the
1316 pS range. Measurement of the single-channel am- Auxiliary subunits of HVA Ca2 channels were first
plitude is complicated by the presence of subconductance identified in purified preparations of skeletal muscle
activity (228). Failure to account for these subconduc- channels (see Ref. 326 for review). This channel complex
tance openings would result in lower estimates, and this was found to contain at least four subunits: 1 (Cav1.1),
may explain why Cav3.2 was reported to have a conduc- 2 (21), (1), and (1). Purified cardiac L-type and
tance of 5.3 pS (90). Subconductance activity has also brain N-type channels have a similar 12 structure, but
been observed with native T-type channels (64). with distinct 1- and -subtypes. Additional members of
The effect of changing external pH has been studied each subunit class have been cloned (Fig. 4), such that
with Cav3.1 (391) and Cav3.2 (95). In contrast to native there are now seven HVA 1-, four -, three 2-, and eight
channels, whose activity is affected by small deviations -like subunits (reviewed in Ref. 171). The -subunits play
from pH 7.2, the recombinant channels are largely unaf- a critical role in channel function, controlling both chan-
fected by pH changes in the 8.2 to 6.9 range. Further nel expression at the plasma membrane, voltage depen-
acidification of the external solution to pH 5.5 decreases dence, and pharmacology. The 2- and -subunits also
the currents measured during standard test pulses. Muta- play a role in expression of functional channels. Muta-
tion of the aspartate in the dmain III pore loop to gluta- tions in Ca2 channel genes, or calcium channelopathies,
mate (EEDD to EEED) shifted the apparent pKa of Cav3.1 have been linked to a variety of disease phenotypes in
from 6.0 to 7.0 (391). In addition,this mutation increased both mice and humans (reviewed in Ref. 248).
the sensitivity to Cd2 block. Therefore, each domain Native T-type channels have yet to be purified, so
contributes a negatively charged residue to form a high- their subunit structure is unknown. Two strategies have
affinity binding site for Ca2 (and Cd2) in Cav3 channels been used to infer its structure: to assay whether anti-
(EEDD) as shown previously for Cav1 and Cav2 (both sense oligonucleotides directed against HVA -subunits
EEEE) channels (75, 207, 319, 449). In addition to affect- can alter native T-type channel function, and to test
ing permeation, shifting the pH from 8.2 to 5.5 produced a whether the activity of the cloned T-type 1-subunit is
dramatic 40-mV shift in the voltage dependence of activa- affected by coexpression with cloned HVA subunits. An-
tion of Cav3.2, lowered its slope factor, and produced an tisense depletion of -subunits causes profound reduc-
11-mV shift in the h curve (95). These results suggested tions in HVA channel density; however, this treatment has
that protonation of the channel affected the voltage de- no effect on T-type channels recorded from the same
pendence of transitions between deep closed states (R to neurons (226, 234). Similarly, coexpression of cloned
C3 transitions in Fig. 3). Although it is unknown where on -subunits has little or no effect on cloned T-type channel
the channel the affected residues lie, they may very well activity (100). In contrast, coexpression with either 21
be in the pore. Consistent with this hypothesis, mutations or 22 was found to double the size of Cav3.1-mediated
in the Cav3.1 pore also lowered its voltage dependence currents (100, 132). Concomitant immunolocalization
and shifted activation to more positive potentials (391). studies supported the contention that this rise in currents

was due to increased expression of 1-21 complexes at A. Antihypertensives

the plasma membrane (100). Similar results were ob-
tained by coexpression of a 22 with mouse Cav3.1 (169), Marketing of mibefradil (Posicor, Roche) as the first
although the effects of 21 failed to reach statistical selective T-type channel blocker stimulated considerable
significance (224). Coexpression with 21 causes no de- interest in the pharmacology and physiology of these
tectable changes in the biophysical properties of Cav3.1 channels (412). Mibefradil was approved for use in essen-
channels, while 22 has been reported to have minor tial hypertension and stable angina pectoris until it was
effects on inactivation (169). Mutations in the murine 22 withdrawn from the market due to drug-drug interactions
gene have been linked to an absence of epilepsy pheno- leading to irregular heart rhythms (219). Submicromolar
type in the mouse strain ducky (23). Recordings from concentrations of mibefradil, formerly Ro-40 5967, pref-
Purkinje neurons isolated from ducky cerebella showed erentially block T-type channels, while higher concentra-
half the P-type current density observed in controls. tions are required to block cardiovascular L-type channels
These results, in combination with coexpression studies, (282). Its 10- to 30-fold selectivity for T- over L-type chan-

suggest that 22 plays a greater role in HVA than LVA nels has been confirmed in a number of preparations,
channel expression. including recombinant channels expressed in mammalian
Identification of the mutation in the mouse strain cells (267, 323). The average IC50 from 11 studies for
stargazer, which also exhibits an absence epilepsy phe- mibefradil block of native T-type channels was 1.5 M
notype, led to the cloning of 2 (233). In silico cloning has (see Table 1 in Ref. 267). Mibefradil potency is affected by
extended the -like family to eight members (78). The the concentration, and choice, of divalent cation, with
2-protein is only 25% identical to skeletal muscle and less block in Ba2 than in Ca2 solutions (267). In 2 mM
may play different physiological roles. Immunoprecipita- Ca2 solutions, mibefradil blocked recombinant Cav3.2
tion studies have shown that 2 may be a subunit of both channels with an IC50 of 0.14 M, whereas slightly higher
HVA Cav2.2 Ca2 channels and glutamate receptors of the concentrations were required for block of Cav3.1 and
AMPA class (74, 190, 366). Coexpression of 2 inhibits the Cav3.3 (267). Mibefradil block of both HVA and LVA chan-
expression of Cav2.1 and Cav2.2 channels in the presence nels is state dependent (41, 278). Single-channel measure-
of 21 and has minor effects on their biophysical prop- ments indicate the presence of open channel block, and in
erties (190, 346). In contrast, 2 had little effect on Cav3.3 addition, a longer lasting block that results in a reduction
expression, although it could slow the decay of the tail in active sweeps (280). Whole cell measurements indicate
current (145). The related isoforms, 3 and 4, have been that inhibition is enhanced by holding the membrane at
reported to have no effect on Cav3.3 currents (145). In potentials where T-type channels inactivate, suggesting
contrast, 2, 4, and 5 have been reported to accelerate the drug has higher affinity for inactivated states. The
inactivation of Cav3.1 currents and shift steady-state in- apparent affinity for these states was estimated to be 83
activation by 4 mV (210). To reiterate, the electrophys- nM using native T-type currents (278), and similar results
iological activity of T-type 1-subunits expressed alone is were obtained with recombinant channels (267). This pro-
very similar to that observed with native channels. This is vides another mechanism for selectivity, since T-type
not the case for HVA channels, which require -subunits channels inactivate near the resting membrane potential
for proper gating (223), and 21- and 1-subunits for of most cells, while HVA channels require depolarization.
proper pharmacological activity (381). In conclusion, A similar mechanism has been invoked to explain the
these studies indicate that HVA auxiliary subunits can selectivity of dihydropyridines for L-type channels in de-
interact with Cav3 1-subunits, but additional biochemical polarized vascular beds over L-type channels in heart.
studies are required to establish this interaction in vivo. These results suggest that mibefradil selectivity in vivo
was probably greater than estimated in vitro since most
V. PHARMACOLOGY studies used Ba2 solutions, and a significant fraction of
T-type channels is inactivated in vivo. Therapeutic plasma
Cardiovascular L-type calcium channels are an estab- concentrations of mibefradil were found to be in the 0.51
lished drug target in the control of blood pressure and M range, with 99.5% bound to plasma proteins such as
cardiac rhythm. Selective Cav1 blockers have been devel- 1-acid glycoprotein (433). Therefore, a significant frac-
oped from over three drug classes, including dihydropy- tion of T-type channels in vascular smooth muscle myo-
ridines, phenylalkylamines, and benzothiazepines. In con- cytes would have been blocked during treatment. Subse-
trast, there are no highly selective drugs or peptide toxins quent studies have established that micromolar
(369) that selectively block Cav3 channels. A comprehen- concentrations of mibefradil are capable of blocking a
sive review of drugs tested on native T-type channels was wide range of ion channels, including those for Na (106),
recently published (158); therefore, this review focuses K (138), and Cl (304). It remains to be determined
on drugs whose mechanism may involve block of T-type whether block of these other channels contributed to its
channels. antihypertensive activity. This ability to block other ion

channels complicates the interpretation of mibefradil ef- B. Antiepileptics

fects, and additional studies are required to establish the
role of T-type channels in smooth muscle proliferation (355), Generalized, or petit mal, absence epilepsies are
cardiac remodeling (113, 422), and renal protection (28). characterized by periods of synchronized neuronal activ-
Akaike et al. (4) have extensively studied the sensi- ity, generating a 3- to 4-Hz spike-wave pattern on the
tivity of neuronal T-type currents to calcium channel electroencephalogram. Considerable evidence suggests
blockers. These studies demonstrated that many drugs that spike-wave discharges are produced by synchronized
blocked neuronal T-type currents at micromolar concen- oscillations of cortical, reticular thalamic, and corticotha-
trations. The order of potency (IC50 in parentheses, in lamic neurons (92, 136, 378). Spike-wave discharges of
M) was flunarizine (0.7) nicardipine (3.5) nifedipine corticothalamic neurons are in part mediated by T-type
(5) nimodipine (7) methoxyverapamil (50) and dilti- channels that produce LTS (93, 277, 377, 387). Ethosux-
azem (70). A similar rank order was found in a study of imide is a first-line drug in the treatment of absence
septal neurons, except nicardipine was found to be more epilepsy (193). In vitro studies found that ethosuximide

potent (IC50 0. 77 M; Ref. 6). Because early studies can block T-type channels with little effect of HVA chan-
using 0.11 M dihydropyridines had shown selective nels in thalamic neurons (86). Because block occurred at
block of L-type channels with little block of T-type chan- therapeutically relevant concentrations (0.3 mM), it was
nels, these authors concluded that there was a subset of concluded that T-type channel block might explain its
dihydropyridine-sensitive T-type channels. A similar con- mechanism of action. Support for this hypothesis came
clusion was reached from a study of thalamic and mes- from the following studies with related analogs: 1) T-type
enteric SMC T-type currents (246, 398). Felodipine is also current block was also observed at therapeutically rele-
a potent T-type blocker, displaying an apparent affinity for vant concentrations of methyl-phenylsuccinimide (MPS),
inactivated channels of atrial myocytes of 13 nM, but only the active metabolite of the antiepileptic drug methsux-
700 nM for those of pituitary GH3 cells (83). Such high- imide, and 2) no block was observed using either the
inactive analog succinimide or the convulsant analog
affinity block led these authors to conclude that block of
tetra-methylsuccinimide (87). With one exception (215),
T-type channels might contribute to felodipines antihy-
most studies have failed to confirm ethosuximide block of
pertensive properties. Amlodipine (Norvasc, Pfizer),
T-type currents at therapeutically relevant concentrations
which is currently the most widely prescribed dihydro-
(403), leading to the suggestion that block of other ionic
pyridine, is 12-fold selective for cardiac L-type channels
channels may be more relevant (232).
(IC50 0.5 M), blocking T-type channels with an appar-
Ethosuximide and MPS are capable of blocking all
ent IC50 of 5.7 M (323). Recombinant Cav3.2 channels
three recombinant T-type Ca2 channels (137). As ob-
were slightly less sensitive (IC50 31 M). Because am-
served with mibefradil, block is enhanced up to 10-fold by
lodipine serum concentrations are 14 nM (432), at least holding the membrane at potentials that partially inacti-
3% of the T-type channels will be blocked during therapy, vate T-type channels. In addition, ethosuximide has
and this fraction might be higher in depolarized cells. greater affinity for open than rested channels, suggesting
The dihydropyridine structure-activity relationships that it is both a gating modifier and an open channel
of T- and L-type channels are different. This is most blocker. The effect of ethosuximide on other recombinant
clearly demonstrated by the stereoisomers of BAY K 8644; Ca2 channels has not been rigorously tested, although
T-type channels are weakly blocked by micromolar con- block of Cav2.3 (IC50 20 mM) has been reported (295).
centrations of both (237), while for L-type channels the In particular, its affinity for partially inactivated HVA
()-isomer is an agonist (198). Stimulation of LVA cur- channels has not been studied, so its selectivity is un-
rents by BAY K 8644 is difficult to interpret because this known. Studies with MPS suggest that it is only about
compound shifts gating of L-type channels to more nega- twofold selective for LVA over HVA channels in thalamic
tive potentials. In contrast, the stereoselectivity and po- neurons (87). The affinity (Ki) of ethosuximide for inac-
tency of niguldipine were similar for both L- and T-type tivated states was estimated to be 2 mM. Because thera-
channels; the racemic mixture blocked T-type currents in peutic plasma concentrations are close to 0.5 mM (57),
guinea pig atrial myocytes with an IC50 of 0.18 M (341). only a fraction of channels is expected to be inhibited
Although the binding site for dihydropyridines is different during therapy. This partial block may be relevant due to
between L- and T-type channels, the mechanism of block pharmacological amplification (297). This is due to the
appears similar. Dihydropyridines appear to stabilize in- role of thalamic T-type channels in gating Na channels,
activated states of both channels, causing a negative shift such that nominal block of the T-type channel is sufficient
in the steady-state availability curve (31, 83, 221, 336). In to block all-or-none action potentials (178).
conclusion, dihydropyridines are selective for L-type T-type channels are also blocked by antiepileptics
channels, and some analogs are capable of blocking T- that are thought to work by blocking Na channels. Per-
type channels in the 110 M range. haps the best studied is phenytoin, which clearly blocks

both channels at therapeutic concentrations (413). Phe- thane, and nitrous oxide block DRG T-type currents at
nytoin appears to be selective for LVA over HVA currents therapeutically relevant concentrations of anesthetics
(413). The mechanisms of block are also similar, with (401, 403). Isoflurane block of either DRG or recombinant
higher affinity for inactivated states as evidenced by its Cav3.1 occurs at concentrations (271303 M) that are
ability to shift the h curve (413). From this shift it can be lower than those achieved during anesthesia (404). Block
calculated that phenytoin blocks inactivated channels is not totally selective, as block of HVA currents by halo-
with a Ki of 7 M. Studies using recombinant T-type thane occurs at similar millimolar concentrations (160,
channels indicated that Cav3.2 was the most sensitive, 390). In contrast, nitrous oxide selectively blocked DRG
although block was variable even in a clonal cell line, LVA currents with no effect on HVA currents (401). In
suggesting that additional factors may be important for addition, nitrous oxide was found to be a selective
block (404). Zonisamide has also been reported to par- blocker of Cav3.2 currents, with little effect on Cav3.1
tially block T-type channels (60% max, IC50 70 M) with currents.
little effect on HVA currents (386). Octanol (20 M), an aliphatic alcohol, was reported

Additional support for the hypothesis that an in- to block LVA currents totally from inferior olivary neu-
crease in T-type currents might underlie epilepsy comes rons (245). Subsequent studies required much higher con-
from animal models. One extensively studied model is the centrations for block (IC50 200 M; reviewed in Ref.
genetic absence epilepsy rats from Strasbourg (GAERS; 158). Recombinant T-type channels can be totally blocked
Ref. 419), whose T-type currents were larger than those by octanol, with 50% inhibition occurring at 160 M for
recorded from a seizure-free rat strain (409). Consistent Cav3.1 and 219 M for Cav3.2 (404). Studies on hippocam-
with the enhanced currents, enhanced expression of pal CA1 pyramidal neurons indicated that octanol is non-
Cav3.2 was detected in the reticular thalamic nuclei, al- selective, blocking T-, N-, and L-type channels to a similar
though the changes in current density (50%) were larger extent. In conclusion, T-type currents are blocked at ther-
than the changes in channel mRNA (20%; Ref. 394). In apeutically relevant concentrations of some anesthetics,
addition, a small increase in Cav3.1 expression in the and this block may contribute to their anesthetic and
ventrobasal nucleus of thalamus was detected at the mes- analgesic properties.
sage level, but not with currents (151). Another interest-
ing result of this study was that T-type channel mRNA was
very similar, and in fact rose slightly, between juvenile (15 D. Antipsychotics
days old) and adult (40 days old) rats. Enhanced expres-
sion of Cav3.2 mRNA was noted at both time points. Antipsychotics are used in the treatment of psychotic
Because the absence epilepsy phenotype of these animals disorders such as schizophrenia, the main phase of manic-
is not manifested until after 30 days, these data suggest depressive illness, and other acute idiopathic psychotic
that maturation of the thalamocortical circuit plays a illnesses (20). Most antipsychotics (neuroleptics) act by
more important role in determining the onset than the blocking D2 class dopaminergic receptors, although drugs
intrinsic properties of these neurons (419, 431). Increased of diphenylbutylpiperidine class can also block T-type
expression of T-type currents has also been found in channels.
hippocampal CA1 pyramidal neurons in three different Among diphenylbutylpiperidines, penfluridol was the
models: a kindling model of focal epilepsy (112), a pilo- most effective at blocking T-type currents of human med-
carpine model of temporal lobe epilepsy (380), and an in ullary thyroid cancer (TT) cells (108). Penfluridol blocked
vitro model (301). These currents, as well as intrinsic with an IC50 of 224 nM, and similar results were obtained
bursting of these neurons, could be blocked by 100 M with adrenal zona fasciculata cells (109). It was also
Ni2 (380), suggesting the involvement of Cav3.2 channels 10-fold more selective for LVA currents over HVA cur-
(230). rents as 500 nM penfluridol inhibited 82% of the LVA
current but only 20% of the HVA. Block of other channels
has not been studied in detail, although it should be noted
C. Anesthetics that these drugs are also potent blockers of K channels
(139) and bind with subnanomolar affinity to L-type chan-
Although the precise mechanism of action of general nels (208). Despite strikingly different structures, thiorid-
anesthetics is unknown, many studies have demonstrated azine, clozapine, and haloperidol have comparable activ-
that they are capable of modulating the activity of ion ity in TT cells, but are still much less active than
channels, including ligand-gated GABA and glycine chan- fluspiriline or penfluridol (108). Similar results have been
nels. A newly described mechanism for inhalation anes- obtained using recombinant rat Cav3 channels, although
thetics is that they hyperpolarize neurons by potentiating the apparent affinity was higher than observed with native
the activity of leak K currents carried by the two-pore channels (349). Cav3.1 channels were blocked with the
domain channels such as TASK-1 (371). Isoflurane, halo- following order of potency (IC50 in parentheses, in nM):

pimozide (43) penfluridol (110) flunarizine (530) afterhyperpolarizations (40, 244, 416). In addition to tran-
haloperidol (1,163). Similar results were obtained with sient increases in intracellular Ca2, T-type window cur-
Cav3.2 and Cav3.3, although flunarizine block of Cav3.2 rents may play an important role in slower increases in
required sevenfold higher concentrations. These com- basal Ca2. This property appears to be important for
pounds all shifted the steady-state inactivation curve to hormone secretion from adrenal cortex and pituitary,
more negative potentials, indicating that they bind pref- myoblast fusion (45), and possibly smooth muscle con-
erentially to the inactivated state. Use-dependent block traction.
has been noted previously in studies of native channels,
suggesting open channels may also have a higher affinity
than rested channels (15, 108). Pimozide binding to D2 B. Future Directions
receptors occurs over the same concentration range
(KD 29 nM) as block of the recombinant Cav3 channels, Cloning of the T-type channel 1-subunits has pro-
suggesting that T-type channels may be blocked during vided tools with which to study the distribution of these

therapy (349). channels. As expected, mRNA for these channels was
found to be distributed throughout the central nervous
system; however, it was very surprising to find expression
VI. CONCLUSIONS
in organs such as liver and kidney. Future studies are
required to discern their role in these tissues. Such stud-
A. Physiological Roles ies would be aided by the development of high-affinity
antibodies. Antibodies would also be useful in studying
The expression of T-type channels in diverse cell the distribution of these channels within neurons and in
types suggests that they play a role in various physiolog- their purification. HVA channels are multisubunit com-
ical functions. The voltage dependence of activation and plexes; therefore, it seems likely that T-type channels will
inactivation provides clues to these roles and places con- also have auxiliary subunits. These hypothetical subunits
straints on when these channels will be active. In neurons may regulate surface expression or might be involved in
where the resting membrane potential is in the 90 to hormonal regulation of channel activity.
70 mV range, T-type channels can play a secondary Because mutations in many ion channel genes have
pacemaker role; an excitatory postsynaptic potential been linked to inherited diseases, it seems likely that
(EPSP) opens T-type channels and generates a LTS, mutations in T-type channel genes would lead to a phe-
which in turn activates Na-dependent action potentials notype. Mutations that lead to enhanced expression of
and HVA Ca2 channels. Therefore, T-type channels play channels, or that reduce inactivation, might tip the bal-
an important role in the genesis of burst firing. In neurons ance to overexcitability. Enhanced expression of T-type
where the resting membrane potential is relatively depo- channels have been observed in animal models of epi-
larized (greater than 70 mV), T-type channels are inac- lepsy (409), neuronal injury (79), cardiac hypertrophy
tivated and an EPSP directly activates Na channels. Due (308), and heart failure (362). The opposite approach is
to their fast recovery from inactivation, T-type channels also being tested by creating transgenic mice that lack
can produce a rebound burst in depolarized neurons fol- expression of Cav3 genes. Studies with the Cav3.1 knock-
lowing an IPSP. Electrophysiological recordings indicate out mouse have established the central role this isoform
that these channels are preferentially localized to den- plays in generating burst firing and thalamic spike-wave
drites, and hence play a role in signal amplification (97, discharges (206). What will be the phenotype of other
256, 265, 328). Large T-type currents have been found in Cav3 knock-out mice?
thalamic neurons, where they play an important role in Somewhat surprisingly the transcriptional regulation
oscillatory behavior. The thalamus acts as a gateway to of Ca2 channel subunits has received scant attention.
the cerebral cortex, and inappropriate oscillations of Preliminary studies indicate that the Cav3.1 promoter is
these circuits, or thalamocortical dysrhythmias, have methylated in some tumors, which presumably leads to
been implicated in a wide range of neurological disorders decreased expression (408). It has been noted that the
(242). T-type currents also appear to play a role in olfac- Cav3.2 gene contains a region with high homology to the
tion, vision, and pain reception (201, 317, 402). consensus sequence of neural restrictive silencer ele-
Calcium influx not only depolarizes the plasma mem- ments, providing another avenue for future research
brane but also acts as a second messenger, leading to the (342). Transcriptional regulation also appears to be im-
activation of a plethora of enzyme and channel activities. portant for hormonal upregulation of T channels (22, 447).
T-type channels can cause robust increases in intracellu- Studies on gene regulation may also provide insights into
lar Ca2, especially in proximal dendrites (294, 460). Cal- the control of T-channel expression during the cell cycle
cium and voltage synergistically open Ca2-activated K (220).
channels, which contribute to spike repolarization and The pharmaceutical industry is using combinatorial

libraries and high-throughput screening to search for new 12. ARMSTRONG CM AND MATTESON DR. Two distinct populations of
calcium channels in a clonal line of pituitary cells. Science 227:
drugs, and hopefully one of these will be a highly selective 65 67, 1985.
T-type channel blocker. Such a blocker might be useful in 13. ARNOULT C, CARDULLO RA, LEMOS JR, AND FLORMAN HM. Activation of
the control of blood pressure, as evidenced by the utility mouse sperm T-type Ca2 channels by adhesion of the egg zona
of mibefradil. If the drug penetrates the central nervous pellucida. Proc Natl Acad Sci USA 93: 13004 13009, 1996.
14. ARNOULT C, LEMONS JR, AND FLORMAN HM. Voltage-dependent mod-
system, it might be useful in a variety of disorders that ulation of T-type calcium channels by protein tyrosine phosphory-
involve thalamocortical dysrhythmias, such as epilepsy lation. EMBO J 16: 15931599, 1997.
and neuropathic pain. Clearly, a highly selective blocker 15. ARNOULT C, VILLAZ M, AND FLORMAN HM. Pharmacological properties
of the T-type Ca2 current of mouse spermatogenic cells. Mol
would also be extremely useful in research and would Pharmacol 53: 1104 1111, 1998.
lead to a greater understanding of the physiological roles 16. ARTALEJO CR, ADAMS ME, AND FOX AP. Three types of Ca2 channel
of voltage-gated Ca2 channels. trigger secretion with different efficacies in chromaffin cells. Na-
ture 367: 7276, 1994.
17. ASHCROFT FM, KELLY RP, AND SMITH PA. Two types of Ca channel in
I thank my current and former fellows Drs. Jung-Ha Lee,
rat pancreatic beta-cells. Pflugers Arch 415: 504 506, 1990.

Juan Carlos Gomora, and Janet Murbartia n for help with record- 18. ASSANDRI R, EGGER M, GASSMANN M, NIGGLI E, BAUER C, FORSTER I,
ings of recombinant channels and proofreading. I thank Jennifer AND GORLACH A. Erythropoietin modulates intracellular calcium in a
DeLisle for countless trips to the library to photocopy articles. I human neuroblastoma cell line. J Physiol 516: 343352, 1999.
thank Drs. John Huguenard, David McCormick, Thierry Bal, 19. AVERY RB AND JOHNSTON D. Multiple channel types contribute to the
low-voltage-activated calcium current in hippocampal CA3 pyrami-
Hee-Sup Shin, Andy Randall, and Chris Benham for permission
dal neurons. J Neurosci 16: 55675582, 1996.
to use figures. I also acknowledge the valuable critiques and 20. BALDESSARINI RJ. Drugs and the treatment of psychiatric disorders.
suggestions provided by my collaborator Dr. Paula Barrett. In: The Pharmacological Basis of Therapeutics (9th ed.), edited by
This work was supported in part by National Institutes of Hardman JG, Limbird LE, Molinoff PB, Ruddon RW and Gilman AG.
Health Grants HL-58728 and NS-38691 and an Established Inves- New York: McGraw-Hill, 1996, p. 399 430.
21. BALKE CW, ROSE WC, MARBAN E, AND WIER WG. Macroscopic and
tigator Award from the American Heart Association.
unitary properties of physiological ion flux through T-type Ca2
Address for reprint requests and other correspondence: E. channels in guinea-pig heart cells. J Physiol 456: 247265, 1992.
Perez-Reyes, Associate Professor of Pharmacology, Univ. of 22. BARBARA JG AND TAKEDA K. Voltage-dependent currents and modu-
Virginia, 1300 Jefferson Park Ave., Charlottesville, VA 22908- lation of calcium channel expression in zona fasciculata cells from
0735 (E-mail: eperez@virginia.edu). rat adrenal gland. J Physiol 488: 609 622, 1995.
23. BARCLAY J, BALAGUERO N, MIONE M, ACKERMAN SL, LETTS VA, BROD-
BECK J, CANTI C, MEIR A, PAGE KM, KUSUMI K, PEREZ-REYES E, LANDER
ES, FRANKEL WN, GARDINER RM, DOLPHIN AC, AND REES M. Ducky
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Physiol Rev

83: 117161, 2003; 10.1152/physrev.00018.2002.

Molecular Physiology of Low-Voltage-Activated

Department of Pharmacology, University of Virginia, Charlottesville, Virginia

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I. INTRODUCTION rapidly ( 1530 ms). The second type peaked at 0 mV

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Electrophysiological studies of recombinant chan- II. ELECTROPHYSIOLOGY OF NATIVE

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TABLE 1. Properties of neuronal low-threshold Ca2 spikes

Resting Deinact V Deinact V Deinact Time Deinact Time

Subthalamic nucleus 50 20 78 80 120 40

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TABLE 2. Properties of T-type currents in isolated neurons

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TABLE 3. Properties of T-type currents in brain slices

Cerebellum 0.5 60 33 690 51M 86 14 100 (19%) 292

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TABLE 4. Properties of T-type currents in peripheral tissues

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TABLE 5. Properties of LVA currents in smooth muscle

Definitions are as in Table 2.

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I. Cell Lines J. Single-Channel Recordings

TABLE 6. Properties of T-type currents in cell lines

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TABLE 7. Single-channel properties

Rat DRG 40 Ca 0.2 5.2 Ca Ba 64

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