Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B.

Parekh
Intracellular Ca2+ Signalling: Ca2+ Influx

Dr. Anant B. Parekh Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, U.K.

Importance of intracellular [Ca2+]

Sperm motility and Fertilisation: LIFE Exocy tosis Muscle contraction

[Ca 2+]

Enzy me activ ity Cell motility Gene transcription Cell growth and prolif eration

BUT: dark side to [Ca2+]: cell death

Necrosis and apoptosis

[Ca2+] is a life or death signal

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

The fact
that an increase in intracellular calcium concentration controls so many processes poses a problem: The calcium specificity problem

The calcium specificity problem

Following a rise in cytoplasmic Ca 2+ concentration, how does a cell know whether it should live or die and, if it is to live, then which Ca 2+-dependent process should it activate?

How can a promiscuous messenger like Ca2+ elicit a specific response?

Calcium specificity problem

Explanations
1. Everything is activated No 2. Different cells are devoted to different tasks No 3. Probable explanation: Intracellular Ca 2+ signals are complex; Ca 2+ signals are often restricted to sub-cellular regions and vary in both space and time Information is encoded in the shape/kinetics of the Ca 2+ signal

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Shape/kinetics of a Ca2+ signal:
balance between the Ca2+ increase and subsequent Ca2+ removal

Calcium increase: opening of calcium channels Calcium remov al: calcium buffers + calcium pumps + calcium transporters

Calcium distribution
[Ca2+ ] 1.5 mM Plasma membrane membrane potential: -70 mV 50 nM

Resting

[Ca2+ ]

E Ca > +100 mV
[Ca2+ ] 1 mM Ca2+ store

Huge electrochemical gradient for Ca 2+ influx

Consequence
of large electrochemical Ca2+ gradient across the plasma membrane

A very powerful way to elevate intracellular Ca2+ is to increase the permeability of the plasma membrane to Ca2+

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Plasma membrane permeability to Ca2+

can be increased by opening Ca2+ channels

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Calcium entry channels

N.B. excitable vs. non-excitable cells
VOCC Mechanical stimulus Ligand-gated

SOC

Ca2+

Ca2+ store

Ca 3 Na Ca 3 Na Ca

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The two major Ca2+-selective channels

Voltage-oper ated calcium channel Excitable cells e.g., nerve and muscle Opened by action potential s Store-operated calcium channel Non-excitabl e cells e.g., immune cells, liver Opened by store depletion SOC

Ca2+
VOCC

Ca2+

plasma membrane

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Techniques
to study Ca2+ channels
1. Electrophysiology: patch clamping 2. Whole cell/perforated patch; excised patch (if possible) 3. Ca 2+-sensitive fluorescent dyes e.g., fura 2 4.
45Ca 2+ uptake

5. Ca 2+-dependent effects: Ca 2+-activated ion channel (Ca-K) Muscle contraction Secretion Most unequiv ocal is patch clamping

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Voltage-operated Ca2+ channels

VOCC

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VOCCs are opened by depolarisation of the plasma membrane, as occurs following an action potential for example Permit rapid and large Ca 2+ entry Can trigger CICR There are several different types of VOCC

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Subtypes of VOCC
High voltage activated

Low voltage activated

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Evidence for sub-types of VOCC

1. Biophysics 2. Pharmacology 3. Molecular biology

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Evidence for sub-types of VOCC

1. Biophysics
A. Current-v oltage relationship
Threshold potential - not true threshold; potential where detectable whole cell current occurs V1/2 (potential where current is half -maximal) Slope (gating sensitiv ity ) Peak current (pA)
Voltage (mV)
-50

Threshold
Current mV -70 -20

+50 Erev

Slope "Threshold potential" V 1/2

T L

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Evidence for sub-types of VOCC
B. Rate of inactivation during a depolarising pulse

T-type channel 100 pA 20 ms 0 mV -80 mV

Rapidly inactivating: brief pulse of Ca 2+ Slowly inactivating: more sustained Ca 2+ increase

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Evidence for sub-types of VOCC

C. Single-channel conductance

High conductance: more rapid and larger local Ca 2+ signal

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Evidence for sub-types of VOCC

2. Pharmacology
Dihydropyridines (nif edipine, nicardipine) are relativ ely selectiv e inhibitors of L-ty pe Ca2+ channels

conotoxin GIVA (f rom cone shell/marine snail) is a relativ ely specif ic inhibitor of N-ty pe Ca2+ channels -agatoxin IVA (f rom f unnel web spider)
is a relativ ely specif ic inhibitor of P/Q-ty pe Ca2+ channels

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Evidence for sub-types of VOCC

3. Molecular biology
Channels are heteromultimers: At least ten

1 , 2, ()

1, 4 and 1 2 genes

N.B. alternate splicing

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Structure of VOCC
Hetero-multimeric channel w ith three main components:

This subunit alone forms a functional VOCC in expressi on systems 1 subunit contains the pore and voltage sensor

2 -

Increases channel expressi on and hence size of current Modifies voltage sensitivity and kinetics of activation and inactivation may affect channel expres sion e.g., skeletal muscle

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Different subunits
have different effects on inactivation
Cell-type specific expression of different subunits can result in distinct VOCCs i.e., great scope for diversity

The same 1 subunit can evoke a larger or smaller Ca 2+ signal depending on the subunit it is linked with; i.e., amplitude/kinetics of Ca 2+ signal could be dynamic

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Topology
of voltage operated calcium channel

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Primary amino acid sequence

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Why are there so many VOCCs?

Does a cell really care about the source of Ca 2+ entry? Put another way, can Ca 2+ entry through different VOCCs evoke different Ca 2+-dependent responses?

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Not all VOCCs
contribute to neurotransmitter release
Spinal i.p.s.cs
-Aga-IVA Remaining fraction of synaptic currents (%) -CgTx Control -Aga-IVA -CgTx Nicardipine

Control

Control

Nicardipine

Hippocampal e.p.s.cs
-Aga-IVA -CgTx Control

Cerebellum Control i.p.s.cs

Spinal cord Control i.p.s.cs

Hippocampus Nicardipine e.p.s.cs 50 pA 10 ms

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Developmental changes
in VOCC function

-Aga-IVA -CgTx -CgTx -Aga-IVA

Cd2+2+ Cd

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Ca2+ -dependent gene expression

is preferentially linked to L-type calcium channels
A B

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Non-channel functions of VOCC

1. Signal transduction 2. Anchoring protein

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L-type Ca 2+ channel in skeletal muscle E-C coupling Skeletal Muscle L-type

channel RyR1 (coupled) RyR1 (uncoupled) V Depolarise

Signal transduction

Cardiac Muscle
L-type channel V Depolarise

RyR2 (uncoupled)

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Anchoring protein
N-ty pe Ca2+ channel at activ e zones

Synaptic Vesicle

Synaptotagmin

Synaptobrevin
Vesicle membrane Inside

Ca2+

Plasma membrane
Outside

Syntaxin

SNAP25

N-type Channel

Ca2+Channel

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Store-operated channels: SOC

Store-operated channels are found primarily in non-excitable cells These channels are opened by the emptying of intracellular Ca 2+ stores (endoplasmic reticulum)

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Store-operated channels:
activated by store depletion

Calcium store

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Store depletion
under physiological conditions is via the second messenger InsP 3
Receptor
G protein PLC

PLC = phospholipase C InsP3

SOC

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

CRAC

Several biophysically distinct store-operated Ca 2+ channels have been described The best characterised and most widely distributed store-operated channels are the CRAC (Ca 2+ release-activated Ca 2+) channels

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Non-excitable cells
CRAC channel
Store refilling AT P production Exocytosis Cell proliferation

Abnormalities in CRAC channels are implicated in human diseases

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Key features of Ca2+ current through CRAC channels ICRAC

Widespread calcium influx pathw ay Activ ation by store depletion; InsP3, SERCA pump blockers like thapsigargin, ionomycin, EGTA Single channel conductance is low (< < 1pS) Implies > 5,000 functional channels per cell Activ ates slow ly Non-v oltage gated conductance Inw ard rectification Highly selectiv e for calcium

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Properties of ICRAC
InsP3
mV 0 -50 +50

CRAC -15 Current pA

-30

-15 -30

pA

50

100

150

200

Time (s)

Activ ates slowly : ~100 seconds to peak

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Very selective for Ca2+

Most selective of all known ion channels Despite Ca 2+ and Na + having similar ionic radii, Na + permeation is undetectable For every 10,000 ions that permeate, 9,999 are Ca 2+

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How is information conveyed

from the stores to the channels in the plasma membrane?

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Simplest model:
Ca2+ release from the stores opens CRAC channels
2. 1. High intracellular Ca 2+ does not activate ICRAC CRACintracellular BAPTAM Ca2+ High 0.2-100 00 activates ICRAC + 2+ does not activate I IP 1. High intracellular Ca CRAC 3 2+ -1-1 3. TPEN Ca 2. High intracellular BAPTA activates ICRAC
-2 3. -2 TPEN -3
-3 00

II CRAC (-pA/pF) CRAC (pA/pF)

100 100 Time (s)

200 200

300 300

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Key point

CRAC channels are activated by the emptying of the intracellular calcium stores It is the fall in calcium content within the store and not the subsequent rise in cytosolic calcium that causes the channels to open

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Three main models

Ca 2+
Store-operated channel

[Ca 2+]

Endoplasmic reticulum

Plasma membrane

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Vesicular fusion model
CRAC channel

Store depletion

Ca2+

InsP3

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CRAC channel Cortical actin

Conformational coupling model

and the rev ised secretion-like conf ormational coupling model
Store depletion Ca2+

InsP3

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CRAC channel

Diffusible messenger model

Store depletion

Ca2+ store InsP3 receptor

X = CIF?
Ca2+

InsP3

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Intracellular Ca2+ Signalling: Ca2+ Influx Dr. Anant B. Parekh

Stromal interaction molecule STIM
RNAi knockdown of STIM1 or STIM2 inhibits ICRAC and store-oper ated influx in several cell types STIM1 is found both in the plasma membrane and the ER membr ane The protein sequence suggests STIM spans the membr ane once The NH2 terminus contains an EF-hand domai n Mutations that lower the sensitivity of the EF-hand for Ca2+ result in constituti ve store-oper ated Ca2+ influx STIM is likely the elusive Ca2+ sensor within the store STIM1 translocates to just below the plasma membr ane following store depleti on STIM has protein-pr otei n interaction domains so it may couple to plasmal emmal proteins

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Some unresolved questions on SOC

1. Specific role of STIM1? Insertion into the plasma membrane? 2. STIM1 partners? 3. Identification of the CRAC channel 4. Selective pharmacological blockers of SOC

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