The mitochondrial permeability transition pore confers

excitability and CICR wave propagation

Andrew M. Oster1, Balbir Thomas2, David Terman1,2, and Christopher P. Fall1,3
1. Mathematical Biosciences Institute, Ohio State University
2. Department of Mathematics, Ohio State University
3. Department of Anatomy and Cell Biology, University of Illinois at Chicago

December 20, 2008

Abstract
Mitochondria have long been known to sequester cytosolic Ca2+ and even to shape intracellu-
lar patterns of endoplasmic reticulum -based Ca2+ signaling. Accumulating evidence suggests
that the mitochondrial network is an excitable medium which can demonstrate Ca2+ induced
Ca2+ release via the mitochondrial permeability transition. The role of this excitability re-
mains unclear, but mitochondrial Ca2+ handling appears to be a crucial element in diverse
diseases as diabetes, neurodegeneration and cardiac dysfunction. In this report, we extend
the modular Magnus-Keizer computational model for respiration-driven Ca2+ handling to
include a transition pore and we demonstrate both excitability and Ca2+ wave propagation
that is accompanied by depolarizations similar to those reported in cell preparations. These
waves depend on the energy state of the mitochondria, as well as other elements of mito-
chondrial physiology. Our results support the concept that mitochondria can transmit state
dependent signals about their function in a spatially extended way.

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 MBI Technical Report: work in progress

Introduction
Calcium (Ca2+ ) plays a critical role in the modulation of intracellular functions as diverse
as fertilization, metabolism and programmed cell death. Furthermore, Ca2+ dysregulation
is implicated in human health problems including diabetes, cardiac disease and Parkinson’s
neurodegeneration. One of the hallmarks of intracellular Ca2+ signaling is the rich dynamic
behavior that has been observed. These range from frequency modulated oscillations in
small cells in response to extracellular hormone to the bistable and excitable spatiotemporal
patterns seen in very large frog oocytes and eggs.
Ca2+ signaling has been a model system for mathematical and computational biology for
years. Numerous theoretical and computational models have explored Ca2+ signaling in the
endoplasmic reticulum (ER) as an oscillatory or excitable system and many of these modeling
studies have involved spatiotemporal simulations which replicate the rich behavior of these
intracellular Ca2+ signals. The fundamental principle involved in the dynamics of ER-based
Ca2+ signaling is so-called Ca2+ -induced Ca2+ release (CICR). This paradigm involves fast
activation and positive feedback onto ER Ca2+ release channels that interact with slower
inactivation to set up a system capable of supporting excitability, oscillations and related
dynamical behaviors. Other factors such as second messenger modulation of channels can
be thought of as excitability parameters or as additional slow variables in the system.
Mitochondria have long been known to sequester cytosolic Ca2+ and even to shape intracel-
lular patterns of ER-based Ca2+ signaling. In a remarkable series of observations, Ichas and
colleagues demonstrated that mitochondria themselves constitute an excitable Ca2+ signal-
ing system that can support waves of Ca2+ release. The central component in this case is
a mitochondrial permeability (MPT) that both releases Ca2+ and is directly or indirectly
responsive to Ca2+ . The positive feedback by Ca2+ on the transition appears to confer
excitability when examined in cell-free experiments.
The MPT has been the nexus of a large experimental effort principally because it appears
that permanent activation of the transition and subsequent depolarization of mitochondria
are an obligate initiating step to most non-receptor mediated programmed cell death. An
extraordinary body of work has led to the biophysical characterization of the properties of
this permeability transition, but to date, the precise molecular players comprising the MPT
remain unknown. It has been hypothesized that the MPT is the result of action of a pore
complex, which has been termed the permeability transition pore (PTP). Players known to
affect the probability of an MPT event are many of the same factors that are implicated in
cell death in both health and disease: Ca2+ , reactive oxygen species, altered membrane lipids,
electron transport chain anomalies and altered gene expression. Computational modeling
of mitochondrial Ca2+ dynamics may therefore aid in the understanding of how to tip the
balance of cell death towards acceleration in the case of cancer therapies or towards protection
in the case of neurodegenerative diseases.
From a theoretical and computational perspective, Ca2+ handling in cells is a complicated
dynamical system comprised of two separate but interacting excitable media: the endoplas-
mic reticulum and mitochondria. Only recently have mitochondria been incorporated into
biophysical models of intracellular Ca2+ signaling and, to our knowledge, only two studies

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 MBI Technical Report: work in progress

have ever considered the PTP. Neither of these models is spatially explicit, and they do
not consider wave propagation. Selivanov et al. first modeled mitochondrial CICR using a
minimal model and replicated their experimental results showing frequency dependence of
PTP opening and excitability. A more recent study by Holmuhamedov et alu̇tilized a much
more complicated model incorporating elements of respiration and other details. This work
is especially interesting because it suggests that mitochondria might parametrically switch
between dynamical modes due to the overall Ca2+ load in the system; however, this study
did not address the phenomenon of excitability.
A primary goal of this paper is to better understand the role of the PTP in the propaga-
tion of waves of depolarization and Ca2+ release seen in experiments [8]. Our approach lies
somewhere between the two previous modeling efforts: We start with the detailed biophysi-
cal model for mitochondrial Ca2+ handling due to Magnus and Keizer [15, 16, 17]and then
incorporate a biophysical mechanism for the PTP, a pH variable similar to those used by
Selivanov et al. [23, 22], and a weak-acid term that is essential for robust mitochondrial
pH-homeostasis. We do not incorporate additional mechanisms or currents, as was done by
Holmuhamedov et al., in order to minimize the computational overhead in our spatial sim-
ulations. These calculations represent the first spatiotemporal simulations of mitochondrial
function of which we are aware. (Note to reviewers: we would be grateful for any assistance
in giving credit to prior work.)
Another goal of this work is to begin to address the complexity of modular models of phys-
iological processes. The modular approach involves incorporating individual mechanisms
tuned separately with biophysical data into a meta-model for a particular system. This
approach is unavoidable if one seeks to perform computational experiments on ever more
interlinked physiological processes; however, the result is extraordinary complexity and of-
ten a less than satisfactory understanding of the underlying mathematical structure and its
properties. Therefore, we have built and tested a relatively complete model of mitochon-
drial Ca2+ handling, including the additional mechanisms related to the PTP, and then we
have begun to reduce this model to the essential elements necessary for excitability and the
propagation of waves of depolarization and CICR. We feel that such collaborations between
computational cell biologists and mathematical biologists will be necessary going forward to
uncover the basis of the complex spatiotemporal phenomena observed in experiments and
replicated in computer simulations.

Biological background: mitochondria as an excitable

medium
Here we will review some of the basic concepts necessary for nonspecialists to understand the
work that follows. It must be emphasized that the field and literature of mitochondrial phys-
iology is vast and quite detailed, and therefore it is impossible to cover this material in any
depth here. We will build from commonly accepted concepts to evidence for mitochondrial
excitability and a hypothesis for the mechanism that we use for our modeling effort.
The first concept is that mitochondria are quite polarized organelles that maintain a elec-

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 MBI Technical Report: work in progress

Outer
Intermembrane Membrane
Space

NADH + Electron Transport

H
Chain
NAD
Mitochondrial
Matrix ADP+P +
H ATP Synthase
ATP
Inner
membrane ADP Adenine Neuclotide
ATP Translocator
Porin

Figure 1: A schematic diagram of basic mitochondrion structure. The outer membrane has large channels (called porin)
making it highly permeable, rendering the intermembrane space to be a close approximation to the cytosol in terms of ionic
concentrations. Within the mitochondria is the matrix. Separating the matrix and the intermembrane space is the innermem-
brane, along which respiration transpires, that is, the chemosmotic process takes place. As such, it is folded into numerous
cristae to greatly increase its surface area.

trochemical gradient (∆Ψ) much like neurons, and the interiors of mitochondria are on the
order of -150mV relative to the cytosol. This gradient is generated by the active pumping
of protons (H+ ) across the mitochondrial membrane by the electron transport chain (ETC)
during aerobic respiration. The generation of ∆Ψ and the dissipation of the proton com-
ponent by the Fo/F1 ATPase to phosphorylate ADP to ATP form the basis of Mitchells
chemiosmotic hypothesis for the transformation of glucose and oxygen substrates to energy
forms usable by cellular processes.
The second concept is that mitochondria sequester and release Ca2+ . Ca2+ introduced across
the plasma membrane or released from the ER is sequestered into mitochondria through the
Ca2+ uniporter, a process driven by the mitochondrial potential, ∆Ψ[21, 7]. Physiological
influx of Ca2+ into the mitochondria causes a measurable concurrent mitochondrial depolar-
ization [13], which indicates a tight coupling of Ca2+ intake to the polarization state of the
mitochondria. There are several means of Ca2+ release from mitochondria. The two princi-
pal pathways of Ca2+ efflux are the Na+ dependent and the Na+ independent transporters
[21]. These two pathways transport Ca2+ against ∆Ψ and thus require the expenditure of
energy; the Na+ dependent Ca2+ exchanger requires the maintenance of an appropriate Na+
gradient and the Na+ independent Ca2+ pump may directly utilize ATP. In the general case,
the sequestration of Ca2+ is quite rapid and the release occurs on a much slower time scale.
Therefore, mitochondria have been considered as damper for cytosolic Ca2+ signals.
The third important concept is that mitochondria exhibit a stereotypic property known as
the mitochondrial permeability transition (MPT). A variety of stressors such as excessive
Ca2+ , reactive oxygen species, shifts in gene expression and other factors have been shown to
cause mitochondria to undergo a catastrophic depolarization. Interestingly, this permeability
transition can occur in two modes: mitochondria in situ have been observed to undergo
transient depolarizations with no apparent ill effect (ichas paper mentions no swelling), and
mitochondria can undergo permanent depolarization which leads to apoptotic cell death.
Because of the known transition inducers and because the permeability transition has been
linked with cell death in a variety of disorders, the machinery associated with the transition
has been described as a sensor of mitochondrial health. One useful analogy is the weighted
regulator for a pressure cooker, which resonates to maintain an appropriate internal pressure

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 MBI Technical Report: work in progress

but which locks open when the pressure is too great. It may be that flickering of the
permeability transition has a physiological role in preventing damage to highly active or
stressed mitochondria. For the purposes here, what is important is that the permanent
activation of the MPT has been shown definitively to open gaping holes in the mitochondrial
inner membrane sufficient to pass anything smaller than 1500DA. Transient opening of the
PTP is thought to be to a low conductance state limited to approximately 350DA. In either
mode ion gradients are dissipated, resulting in the rapid release of mitochondrial Ca2+ into
the cytosol.
What we have discussed thus far is reasonably accepted. Going forward as we build and
explore our model, we move from consensus to hypotheses with somewhat less experimen-
tal support. For example, a central problem in the permeability transition field is that a
channel has not been definitively isolated, although it may be the same as the electrophysio-
logically characterized mitochondrial megachannel. A functional pore instead of some phase
transition or other phenomenon in the membrane is most likely and it has been termed the
mitochondrial permeability transition pore (PTP, as opposed to MPT). It is a matter of
further controversy as to whether the pore might comprise a single channel or a complex
of proteins. Consistent with the transition physiology, this pore may have two open states
sensitive to different stressors which result in a transient, low conductance state (PTPl ) and
an high conductance state with irreversible opening (PTPh ).
Mitochondria have been demonstrated in vitro to be Ca2+ - excitable organelles capable of
supporting CICR, and there is supporting evidence from in-situ recordings. The work we
describe now is central to our interest in this problem, and the resulting hypotheses are
central to our computational investigations. The key in vitro evidence comes from work by
Ichas, Holmuhamedov, Mazat and colleagues. They were able to quantitatively demonstrate
Ca2+ induced excitability in isolated mitochondrial suspensions, and, in an elegant experi-
ment using mitochondria embedded in a planar gel, to demonstrate that mitochondria can
propagate a wave of CICR. They were further able to demonstrate that mitochondrial CICR
(mCICR) is blocked by cyclosporin A (CsA), which implicates the PTP. In situ, mitochon-
drial flickering and coordinated depolarizations have been shown to depend on both Ca2+
and the PTP. In the Xenopus oocyte, which is large enough for the visualization of complex
spatiotemporal Ca2+ waves, energization of mitochondria has been shown to amplify rather
than dampen ER-based Ca2+ waves, consistent with increased excitability. Such a role may
help to explain the close apposition of the extended syncytial webs of endoplasmic reticulum
and mitochondria in cells.
Holmuhamedov et al. attribute mCICR to PTPl . Notably, PTPl is not thought to be directly
Ca2+ sensitive. Instead, in their model, pH is the initiator of PTPl opening. Ca2+ rushing
in through the Ca2+ uniporter results in a depolarization, which then causes the electron
transport chain to compensate. Heavy ETC activity results in an increase in pH, which
triggers the PTPl . Excitability implies a threshold effect, and, in this case, the threshold is
determined by the ability of weak acidic inorganic phosphates to buffer the pH and compen-
sate for ETC activation. Thus large or repetitive cytosolic Ca2+ transients can overwhelm
pH buffering and cause PTPl activation, while the ETC response to low or slow cytosolic
Ca2+ transients is adequately compensated for.

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 MBI Technical Report: work in progress

(a) (b)
80 60 s 60 s 60 s
60 s max
2+
Ca C 40 Ca
(µM)
∆Ψ

0
min
170

∆Ψ 150 max

90
Ca C
7.65

7.64 min
pH

7.62 With CsA

180 s 180 s

Ca M

∆Ψ Ca C

Figure 2: Key experiments, performed by Ichas et al. [8], demonstrating mitochondrial excitability and its dependence upon
pH (redrawn from [8]). In (a), three pulses of Ca2+ are introduced at different rates with the same amount of Ca2+ introduced.
When Ca2+ is introduced at 30s intervals, the Ca2+ is efficiently sequestered away in the mitochondria, leading to a rise in
the mitochondrial calcium load. The pH rises transiently, yet returns to a state of homeostasis. When the Ca2+ is introduced
at 10s intervals, the pH rises precipitously and this corresponds with PTPl . The pH then rapidly drops, the innermembrane
potential drops, and Ca2+ leaves the mitochondria and enters the cytosol. The Ca2+ is slowly reabsorbed by the mitochondrion
as the pore closes, so that the system eventually returns to a homeostatic state. In (b), a bulbous of calcium is introduced in a
gel containing isolated mitochondria that initiates traveling waves of Ca2+ and potential. By previously dousing the gel with
cyclosporin A (CsA), an agent which obstructs pore opening, both the Ca2+ and potential changes do not propagate the length
of the gel. We suggest that wave propagation is due primarily to diffusion, however the pore itself is necessary for continued
propagation.

A model of respiration driven mitochondrial Ca2+ handling

with a PTP module

Modeling background
There are two principal areas in which Ca2+ in relation to mitochondria have received theo-
retical treatments. One is in the context of ER based Ca2+ release in the cytosol, and these
models have been presented with varying degrees of biophysical detail. Beginning in 1998,
Marhl, Schuster and colleagues have explored the consequences of mitochondrial function on
intracellular Ca2+ signaling using a phenomenological model of mitochondrial Ca2+ handling,
arguing that mitochondria may serve to normalize the amplitude of intracellular Ca2+ os-
cillations [19]. This initial work was followed by a similarly phenomenological, but spatially
explicit, model of ER-mitochondrial Ca2+ handling in the Xenopus oocyte [18] which sought
to explore the paradoxical increase in amplitude and frequency seen in periodic Ca2+ signals
with enhanced mitochondrial function [10]. Fall and Keizer combined a reduced ER Ca2+

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model with a biophysical mitochondrial Ca2+ handling model (discussed below) to show that
mitochondria move the combined dynamics of Ca2+ signaling into an oscillatory mode under
certain parameter regimes.
The other area is models of mitochondria themselves focused on Ca2+ handling and its conse-
quences. The pioneers in this area were Magnus and Keizer, who developed a quite detailed
biophysical model of mitochondrial Ca2+ handling and, in a series of papers [15, 16, 17], ex-
plored the role of mitochondria in pancreatic β-cell Ca2+ oscillations. This work has served
as an important basis for several subsequent efforts to examine the role of Ca2+ stimulation
of mitochondrial metabolism at ATP production and the interplay of the mitochondria with
ER Ca2+ signaling [4, 3, 20]. Two efforts bear more directly on the role of the PTP in mCICR
that we explore here. The first model to explore the role of the PTP was a minimal model
by Selivanov, Ichas, Holmuhamedov, Mazat and colleagues which replicated their mCICR
experimental findings discussed above [23]. Holmuhamedov and colleagues followed up on
this work with a far more biophysical model that used elements of the Magnus and Keizer
model along with additional mechanisms such as a K+ flux and changes in mitochondrial
volume. The authors proposed that the cellular Ca2+ load can serve as a bifurcation param-
eter for the two MPT behaviors (transient PTPl and sustained PTPh ) and demonstrated this
compelling hypothesis in their model which included a a two state PTP mechanism.
We were faced with choosing between the Magnus-Keizer model and the Holmuhamedov
model as the basis of our investigations and we have chosen the more accepted Magnus-
Keizer model for several reasons. For example, volume changes, which are included in the
Holmuhamedov model, do not appear to be relevant to PTPL and wave propagation. In
addition, our goal is to arrive at a more minimal model. Finally, we are interested primarily
in excitability.

The modular Magnus-Keizer model

The Magnus-Keizer model forms that basis of our exploration of depolarization and wave
propagation, and here we briefly describe only those details of the model that will be used
in our study. We note that the original description of the Magnus-Keizer model required
three separate papers and these papers were condensed from a nine volume dissertation by
Magnus [14]. For a description of the individual mechanisms, we refer the reader to the
original papers.
The full Magnus-Keizer model describes details of mitochondrial Ca2+ handling and ATP
production, and includes respiratory proton pumping, ADP phosphorylation by the Fo/F1
ATPase, a proton leak, the Adenine Nucleotide Transporter, the mitochondrial Ca2+ uni-
porter and the associated Na/Ca2+ exchanger. Metabolic pathways and controls are also
explicitly modeled, including glucose-dependent glycolytic and TCA-cycle production of
NADH (under Ca2+ control), glycolytic phosphorylation and hydrolysis of ATP. Finally,
metabolic function is linked to membrane behavior with plasma membrane Katp channels
interacting with voltage gated Ca2+ and K+ channels. The resulting system of 12 dynamic
variables captures the bursting behavior of the pancreatic β-cell in response to various levels
of glucose.

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 MBI Technical Report: work in progress

In our version of the original Magnus-Keizer model, we have eliminated the plasma mem-
brane mechanisms. We have also incorporated two modifications that have been published
previously [4]: First, the uniporter mechanism was reverted to an earlier formulation that al-
lows for saturation in Ca2+ uptake [7]. Second, the scaling and compartmentalization scheme
was modified so that this modular model could more easily be incorporated with existing
Ca2+ signaling models. With these choices of parameters, the dynamics of the original model
are unchanged.
A complete description of the differential equations model used in this paper is given in the
Appendix. We have slightly altered the notation used in the previous work of Magnus-Keizer
for clarity, and use J’s to represent fluxes; however, superscripts now track the type of ion
or proton being trafficked and subscripts signify the process or channel through which the
Ca
flux passes. For example, Juni represents the calcium flux through the Ca2+ uniporter.
In order to study the role of the PTP in excitability and wave propagation, we have added
several features to the Magnus-Keizer model. These include a mechanism for the PTP based
on the hypothesis of Ichas and colleagues. Because the PTP relies upon dynamic pH, which
was constant in the Magnus Keizer model, we have also included a mechanism to track pH.
Finally, we have incorporated an electroneutral inorganic phosphate flux term which serves
as a weak acid as described by Ichas and colleagues. This flux provides for a mechanism
of mitochondrial pH buffering and allows the mitochondrial proton concentration to return
to homeostasis. These additional mechanisms are described in detail in the following sub-
sections. In Figure 3, we show the structure of the Magnus-Keizer model with additional
components colored in blue.

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 MBI Technical Report: work in progress

Mitochondrial
Matrix Cytosol

H+ Respiration
H+
oxidation

NADH NAD +

reduction

Proton Leak H+

Weak Acid H+

PTP H+

H+
F0F1 ATP-ase
F0F1 ATP-ase
ADP
ATP ADP
TCA cycle

Adenine Nucleotide ATP 4- hydrolysis

Translocator
3-
ADP ATP ADP + P

glycolysis

Ca 2+
Uniporter Ca2+

Na Exchanger Ca2+
+
Na

Ca2+
PTP

Figure 3: Schematic of the model currents and compartments. The mitochondria compartment is colored in green and the
cytosol in yellow. Arrows show fluxes and processes relating to H+ , Ca2+ and ADP, which are displayed in the three dashed
boxes, and each process is tracked by a differential equation. Additions to the Magnus-Fall-Keizer model are colored in blue
and the Ca2+ current from the pore shows arrows in both direction as Ca2+ , yet Ca2+ being ejected from the mitochondrion
to the cytosol is particularly of interest.

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Elements of the mitochondrial permeability transition pore model

Our model for the hypothesized PTP is based on the properties outlined by Ichas and
colleagues [9]. Briefly, the PTP displays three states: a closed state, a low-conductance open
state (PTPl ), and a high-conductance open state (PTPh ). Evidence by Ichas [8] suggests that
the PTP enters the low-conductance state in a pH dependent manner, see Figure 2(a). The
transition of the pore to the high-conductance state requires prolonged levels of mitochondrial
Ca2+ , and once the pore has opened to this state it remains open, ultimately leading to cell
death.
H+
Cytosol

H+
H+
H+
H+
2+
PTPl
Cytosol Mito Ca

Low Conductance State

pH M
2+
Ca 2+
2+ Ca H+
Mito Ca Cytosol

Closed Ca M

Mito
Ca
2+
PTPh
High Conductance State

Open
Figure 4: The Ichas-Mazat model of the PTP: closed, PTPl and PTPh . The transition to the low-conductance state depends
upon the pH in the matrix and the transition to the high-conductance state depends on the mitochondrial Ca2+ load. With
pore opening, protons and Ca2+ rush into the mitochondria, dissipating ∆Ψ, and a transition occurs leading to the expulsion
of Ca2+ into the cytosol.

In this section, we outline new additions to the Magnus-Keizer model including the currents
flowing through the PTP, the contribution of the weak acid flux, and the mitochondrial
matrix proton dynamics. The pore gating variable, P T Pl , in its low and high conductance
state will determine if the PTP is open or not.
We assume that protons and Ca2+ travel through the pore while in its open states, as would
other ions not considered in this model. The proton flux is taken to be of the Goldman-
Hodgkin-Katz form multiplied by the state of the pore:
!
−F ∆ψ
X exp( )
JPHT P = φH
i P T Pi ∆ψ HM − HC
RT
−F ∆ψ
(1)
i=ℓ,h
1 − exp( RT
)

where i = ℓ, h indicate whether the pore is in the low or high conductance state with an
associated permeability of φi .
For consistency, the Ca2+ flux is taken to be proportional to the flux through the uniporter

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 MBI Technical Report: work in progress

yet dependent upon the state of the pore.

JPCaT P = κCa
P T P P T Pl Juni (CaC , CaM ) (2)

where CaC and CaM denote cytocolic and mitochondrial Ca2+ levels, respectively, and the
constant κCa
P T P is given in the Appendix. Alternatively, other models (e.g., [23, 22]) take the
uniporter and flow through the pore to be a modified Goldman-Hodgkin-Katz flow with a
correction term.

A module for the weak acid flux

Here we describe an electroneutral inorganic phosphate flux (Π) which serves as a weak acid
and provides a mechanism for mitochondrial pH buffereing. There is little easily applicable
data on the dynamics of Π and pH buffering on which to base a modular mechanism. Seli-
vanov [23] took the weak acid flux to be linear; however, our computational studies suggest
that a linear weak acid flux is not sufficiently robust to account for observed excitability and
wave-like dynamics. Pokhilko et al. [22] developed an extensive model of Π which involves
several differential equations and accounts for phosphate levels within both the matrix and
the cytosol. Their model treated the weak acid flux as a hydroxide(OH− )/phosphate ex-
changer which exported hydroxide from the matrix. Due to basic chemistry, we remind the
reader that
pH + pOH = 14. (3)
So that a decrease in OH− would be tied to a rise in H+ . Over a wide range of parameters,
the weak acid flux due to Pokhilko et al. effectively reduces to a sigmoidal function of pH. We
have, therefore, adopted a simplified sigmoidal form for this restorative flux of pH buffering.
It is unclear what occurs to the weak-acid flux during a pore opening event. One could
account for such behavior by introducing an nonlinearity to the the Michaelis-Menten-like
weak acid flux or we could consider a very ”sharp” weak acid flux as shown below in Figure
, which would turn off when the pore opens and the pH drops below its usual homeostatic
value. homeo −H ) !
(HM M
H 1 + tanh( 10−σ
)
Ja (HM ) = kah (4)
2
homeo
with kah = 70, HM to correspond to a pH value of 7.619, and σ is a measure of the
steepness of the weak acid flux.

A module for proton dynamics

In the Magnus-Keizer model, mitochondrial pH was not considered to be a dependent variable
and was assumed to be at steady-state. Because we are basing our investigations on the
premise that PTPL is pH gated, we have had to add a differential equation to track changes
in mitochondrial pH. The Magnus-Keizer model contained three H+ fluxes: respiration, the
FoF1 ATPase, and a ψ-dependent leak term. To these we have added the H+ flux through

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 MBI Technical Report: work in progress

nmol / [ (mg−protein)(min) ]
80

60

40

20

0
7.65 7.63 7.61 7.59
pH

Figure 5: Phenomenological weak acid component that becomes stronger in a sigmoidal fashion when the mitochondrial pH
is disrupted from its homeostatic value. The strength of this proton flux is on the order of one fourth the size of the dominant
proton flux due respiration.

the PTP and the electroneutral weak acid flux Π. Hence, we can write a differential equation
for changes of the mitochondrial proton concentration as
 
dHM fHM M
= (JLH + JFH0F 1 − Jres
H
+ JaH + JPHT P ), (5)
dt VM τmin

where fHM is the fast buffering constant for protons within the mitochondria. Note that the
current due to respiration and the ejection of protons out of the mitochondria has a different
sign than the other currents (proton leak, F0F1 ATP-ase, weak acid, and the proton current
through the pore).

A module for channel representation of the PTP

We model the opening and closing of the pore in a standard Hodgkin-Huxley-like fashion.
Denote the number of channels that are in the closed, low-conductance and high-conductance
states as P T Pclosed, P T Pℓ and P T Ph , respectively. We assume conservation so P T Pclosed +
P T Pℓ + P T Ph is constant. The dynamics of the low-conductance state is taken to depend
upon pH and is governed by the equation

dP T Pℓ [P T Pℓ∞(Hm) − P T Pℓ ]
= (6)
dt τℓ (Hm)

where both the opening rate, P T Pℓ∞, and the time constant, τℓ , depend upon the mitochon-
drial proton concentration Hm, i.e., mitochondrial pH. In particular, we take
  
p1 − u
P T Pℓ (u) = 0.5 1 + tanh
∞
(7)
p2

and
p5
τℓ (u) = + p6 (8)
cosh( (u−p 3)
(2p4 )
)
where the constants pi are given in the Appendix. We display P T Pℓ∞ and τℓ in Figure .

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 MBI Technical Report: work in progress

5
x10
0.8 10

0.6
PTPl
0.4 5

0.2

0 t=0
0

7.61 7.62 7.63 7.64 7.65 7.66 7.67

pH

Figure 6: The equilibrium values of the low-conductance open state of the pore as a function of pH with the associated
time constant for the low-conductance state. Superimposed upon this is a trajectory from a triggered pore opening. Note that
once the pore opens, the pH is rapidly reduced due to the influx of protons, thus the gating variable does not reach its maximal
value of 1.

Results

Addition of the PTP confers excitability to the Magnus-Keizer

model
A critical proof of concept of our introduced model is the reproduction of the dynamics
outlined by Ichas et al. [8] where the low-conductance state of the PTP was induced by
rapidly stimulating with Ca2+ . The pore flickered depending upon whether the pH of the
mitochonodrial matrix, which rises transiently with Ca2+ stimuli, was sufficiently high. The
pore does not flicker and the mitochondrion returns to homeostasis if the simulation is at
a low frequency. To replicate this behavior, we numerically simulate the system using a
protocol which stimulates the cytosol with three Ca2+ pulses for a duration of 2s over a
period of 25s and 90s. During slow stimulation, i.e., 90s stimulation protocol, the pH rises
transiently but ultimately returns to a homeostatic value, slightly elevated in relation to its
beforehand value since Ca2+ is conserved. However, with the faster stimulation, the pH rises
above a threshold and the pore flickers, that is, the pore transitions to its low-conductance
state.
In its open state, protons rush into the matrix, rapidly returning the pH to its homeostatic
value. In effect, PTPl acts as a pressure release where the cell rescues the ’proper’ proton
distribution. During this rapid return to pH homeostasis, the potential across the innermem-
brane has dramatically dropped. With this lower potential difference, the Ca2+ uniporter
ejects rather than takes in Ca2+ . Likewise, when the pore is open and the potential difference
is reduced, Ca2+ is expelled through the pore.
It is evident that the potential drop induces the the expunging of the Ca2+ from the mito-
chondria by following the trajectories in time, as shown in Figure , where with pore opening

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 MBI Technical Report: work in progress

Pore Remaining Closed Pore Flickering

6 6
5 5
4 4
Ca C 3 3
2 2
1 1
0 0
0 100 200 300 0 100 200 300

170 170

160 160

150 150
ΔΨ
140 140

130 130

120 120

0 100 200 300 0 100 200 300

7.68 7.68

7.66 7.66
pHm
7.64 7.64

7.62 7.62

0 100 200 300 0 100 200 300

7 7

6 6

5 5
CaM
4 4

3 3

2 2

0 100 200 300 0 100 200 300

1 1

0.8 0.8

0.6 0.6
PTP
0.4 0.4

0.2 0.2

0 0
0 100 200 300 0 100 200 300

Figure 7: A simulation demonstrating that the model captures the transient PTP behavior as in [8]. By stimulating with
three pulses of Ca2+ over a span of 90s, no pore opening occurs, yet when one stimulates with the same amount of Ca2+ in the
shorter span of 25s, the pore gating variable for the low conductance state becomes non-zero. See Figure 2 to compare with
the experimental observations.

in (a) the potential rapidly drops and in (b) we note that when the potential is sufficiently
low and the pore is open, cytosolic Ca2+ dramatically rises.
The Ca2+ pulses cause for a rise in the cytosolic Ca2+ concentration and in turn change the
potential difference across the inner membrane via flow the the Ca2+ uniporter. Changes
in ∆ψ then affect the proton fluxes, as evident in Figure . The proton flux that undergoes
the most dramatic change is that associated with the ATP-ase. It precipitously falls with
decreases of ∆ψ, in turn H+ falls, which is equivalent to a rise in pH. Associated with this
period, the mitochondrion expels protons through the ATP-synthase to restore the electro-
chemical gradient. While in this state, the mitochondrion relies solely on the TCA cycle
to produce ATP. The rapid changes in the pH due to the Ca2+ pulses, lead to the pore
transitioning to it’s low-conductance state, which allows a large influx of protons through

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 MBI Technical Report: work in progress

Pore Remaining Closed Pore Flickering

14 14
12 12
10 10
ADPm
8 8
6 6
4 4
2 2
0 0
0 100 200 300 0 100 200 300

2 2

1.5 1.5

1 1
ADPc

0.5 0.5

0 0
0 100 200 300 0 100 200 300
5 5

4 4

3 3
NADHm
2 2

1 1

0 0
0 100 200 300 0 100 200 300

Figure 8: Secondary variables during stimulation.

t=0

160 7
150 6
5
140
∆Ψ 4
130
Ca C
Pore
3
Opening
120
2
Pore t=0
110 1
0 Opening
0.2 00
0.4
7.61 0.2
PTPl 0.6 7.62 0.4 7.62 7.61
7.63 7.63
0.8 7.64 0.6 7.64
7.66
7.65 PTPl 0.8 7.65
1 7.67 pH 1 7.67 7.66 pH

Figure 9: Trajectories of flickering event with the initial point labeled and an arrow indicates the direction of the trajectory.
The x-axis and y-axis are the gating variable P T Pℓ and mitochondrial pH, respectively. In (a) we plot on the z-axis the potential
ψ and in (b) the z-axis tracks the cytostolic Ca2+ concentration. If no Ca2+ is applied, the plot would remain at its initial point
in the upper left hand corner. We infuse the cytosol with three Ca2+ pulses. Each Ca2+ pulse drops the potential ∆ψ, seen in
(a), and an upswing in the cytostolic calcium, shown in (b). After the first two pulses, the potential recovers to approximately
its initial value, but the pH has risen, i.e., [H+ ]M has dropped. During the third Ca2+ pulse, the pH is sufficiently high to
elicit a PTPl event. The pore opens. H+ rushes into the mitochondria causing the potential to drop and in turn the uniporter
ejects calcium into the cytosol, demonstrated in (b).

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 MBI Technical Report: work in progress

the pore (i.e., the JPHT P flux). This greatly lowers the membrane potential, ∆ψ, and causes
the uniporter and pore to expel Ca2+ from the mitochondrial matrix, resulting in the wave-
crest of cytostolic Ca2+ pictured in Figure . In effect, the mitochondria can dampen ’slow’
Ca2+ signals and also amplify ’fast’ Ca2+ signals by this mechanism. We demonstrate nu-
merically that this outward rush of Ca2+ can lead to the emergence of a traveling wave in
the potential and in the cytostolic Ca2+ .

300

Jhres
250

Jptph
200

Jhf1
150

100

Jah

50
Jhl

0 50 100 150 200

Figure 10: H , is the only outward flux of protons from the

Proton fluxes leading to PTPh . The flux due to respiration, Jres
matrix. Note that at each time the cytosol is stimulated with calcium, the flux of protons through the ATP-synthase, JFH0F 1 ,
dramatically drops, suggesting that the changes in this flux are the principle reason for the drops in mitochondrial proton
concentration (and dually the rises in the matrix pH) which occur with each application of Ca2+ . After time t = 125, the pore
opens and a flood of protons enter the matrix through the PTP and help to return the mitochondrion to pH homeostasis. This
influx of protons destroys the electrical potential and the ATP-synthase actually ejects protons until the potential eventually
recovers (hundreds of seconds later.

Cytosolic glucose level affects mitochondrial excitability

The Magnus-Keizer model included a treatment of the metabolic pathways and controls,
including glucose-dependent glycolytic and TCA-cycle production of NADH (under Ca2+
control). The resulting model captures the dynamic behavior for various levels of glucose.
In the Magnus-Keizer model, the glucose levels is treated as a parameter. The amount
of glucose directly affects the availability of NADH, the principle substrate of respiration.
That is, a low glucose level corresponds to a low level of NADH and a high glucose level
is associated with a high level of NADH. Since NADH levels play an integral part of the
respiration process, the glucose level affects the mitochondrion’s respiration capability. As
such, since changes in the pH occur due to increases in the respiration, a mitochondrion’s
degree of excitability also is tied to the glucose level. We consider the same parameters as

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 MBI Technical Report: work in progress

previously used in the above simulation, but now vary glucose. We find that for lower values
of the glucose levels, the pore opening event is diminished.

glc=1.0 glc=2.0 glc=3.0

6 6 6
5 5 5
4 4 4
Ca C 3 3 3
2 2 2
1 1 1
0 0
0 50 100 150 200 250 300 350 0
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350

Figure 11: In a), we perform the Ca2+ stimulation procedure to induce flickering event with a set glucose level of mM.
We note that no pore opening occurs and the system returns to approximately the same steady-state. In b), we repeat the
Ca2+ stimulation procedure, but with a higher level of glucose (2mM). We found a pore opening event but to a lesser extent.
When we raise the glucose level to 3mM, the Ca2+ stimulation procedure induces a large pore opening even and there is a
dramatic out rush of Ca2+ into the cytosol.

Impaired pH buffering can lead to PTP oscillations

The pH buffering capability in the matrix is in part due to the transport of phosphate
carriers across the inner membrane. This effective flux of protons into the matrix is termed
the weak acid flux. The precise form of the weak acid flux is not well understood, so we
consider a phenomenological, sigmoidal representation of the weak acid flux that depends
upon the pH of the matrix. We consider a range of shapes for the weak acid flux that vary
in steepness and in absolute magnitude. For ”strong” and sharp weak acid fluxes, we find
that the mitochondrial dynamics go to a stable steady-state. However if we weaken and/or
take a less sharp representation of the weak acid flux, that is, we reduce the parameter
σ in Equation 4, oscillations in the matrix pH, calcium level, and potential arise. These
oscillations are due to a periodic opening (”flickering”) of the PTP and differ are from the
oscillations as studied by Bertram et al. where the energetics enter into oscillations. In our
model, the recovery pathway of the weak acid flux is not sufficiently strong to keep the pH
at a healthy level. Instead the pH rises above the threshold and the pore transiently opens
in order to regulate the pH level.

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 MBI Technical Report: work in progress

80
σ=5

σ=2

nmol / (mg-prot min)

60
σ=1.5
40

20

6.5 7 7.5 8 8.5 9

Figure 12: By decreasing σ in Equation 4, the strength and sharpness of the weak acid flux changes in kind. Here we plot
three representative plots with a fixed kah but with σ taking on the values of 5,2, and 1.5.

a) b) c)
σ=5.0 σ=2.5 σ=1.0

180 180 180

ΔΨ ΔΨ ΔΨ
80 80 80
0 0.025 0 0.025 0 0.025

PTP 1 0.021
Hm
PTP 1 0.021 Hm PTP 1 0.021 Hm
Figure 13: By decreasing σ the strength and sharpness of the weak acid flux also decrease. In a), σ = 5 and the weak acid
flux is strong and sharp. There exists a stable steady state with the PTP remaining closed. When σ is decreased to σ = 2.5,
the stable steady of the pore remaining closed is lost, with the pore opening and then relaxing to a permanently open state,
pictured in b). However if σ is furthered decreased to a value of 1.0, the trajectories enter into an oscillatory state and there
are oscillations in pH, Ca2+ , potential, and the other dynamic variables (not all pictured).

The mitochondrial excitable medium supports a traveling wave

In a preparation using mitochondria isolated from Ehrlich cells and suspended in a gel, Ichas
et al. found traveling depolarization and Ca2+ waves [8]. This experiment demonstrated that
mitochondrial populations constitute an autonomous Ca2+ -excitable medium for propagating
electrical and Ca2+ signals in space [8]. The most obvious source of spatial coupling in the
system is diffusion of Ca2+ in the gel. We extend our model to describe this behavior by
extending the system in space (one-dimension) and include a term for the diffusion of Ca2+ in
the gel. In this section, we use the cytosol notation for the gel, yet take into account the
gel’s lack of buffering capability.
In the case of a gel, no buffering is present. We can explore the effects of buffers on the
propagation of a wave by considering an effective diffusion rate of unbound Ca2+ with no

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 MBI Technical Report: work in progress

mobile buffers, which is reasonably approximated (see Equation (24) in [24]) by

KC
Def f ≈ DCa = fC DCa (9)
KC + BT
where KC is the disassociation constant for binding and unbinding of Ca2+ to stationary
buffers and BT is the total concentration of stationary buffer binding sites [24]. Typical
measured values for fC range between 0.01 and 0.05 (See chapter 5 of [5]). Since the con-
centration gradient is large when a calcium stimulus is applied, we include a diffusive term
for cytostolic calcium using Fick’s law, so that our dynamics for cytosolic calcium are
 
∂ ∂ 1 Ca Ca Ca Ca
CaC (x, t) = fC DCa 2 CaC (x, t) + [M (Jna,ex − Juni − JP T P ) + Jstim ] (10)
∂t ∂x Vc τmin
where fC = 1 in the case where the mitochondria are suspended in a gel with no calcium
buffering. In the cytoplasm, the diffusion constant of unbound Ca2+ has been estimated to
be about 225-300µ2/s [1, 25].
Without the inclusion of the permeability transition pore, it rapidly diffuses until it is ab-
sorbed by the underlying mitochondrial network. However, with the inclusion of the PTP,
we find excitable behavior: traveling waves of calcium and potential that can propagate
the length of the domain or in certain instances become damped and eventually become
extinguished.

CaC ΔΨ
250 250

200 200

150 150
t
100 100

50 50

0 0
0 50 100 150 200 0 50 100 150 200
x x

Figure 14: A 5 micron strip at x = 0µ is stimulated at t = 15s with a bulbous of Ca2+ . The Ca2+ rapidly diffuses in
the gel until the majority of it is absorbed by the underlying mitochondrial network. The diffusion constant for calcium is set
at DCa = 250µ2 /s. We show the results of a simulation with the PTP included and a mitochondrial proton buffering rapid
buffering coefficient of fHM = 1.8x10−6 . At x ≈ 50µ, the speed of propagation drastically changes; diffusion has run its course
and the pore flickering excitability propagates the wave, but at a slower rate. We plot cytosolic Ca2+ , with an altered color scale
where all values over a concentration of 2µM are truncated. We also plot the potential difference across the innermembrane.

We find that wave propagation depends on the rapid buffering constant, fHM (See Equation
5). For strong proton buffering in the matrix, Ca2+ rapidly propagates at a speed approx-
imately that of diffusion in the gel, but ultimately dies as the calcium is absorbed by the
mitochondrial network (See below ).

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 MBI Technical Report: work in progress

However, the experiments by Ichas et al. suggest that the wave propagates at a rate slower
than diffusion in the gel. In the span of 180s the potential and calcium wave travels ap-
proximately 250µ. The speed of propagation is slower than 1µ/s, that is, the wave speed
was not determined by the rate of diffusion of free calcium in gel, rather was limited by the
dynamics of the mitochondrial network. With the inclusion of the permeability transition
pore and weaker proton buffering, the rapid influx of Ca2+ results in continual pore opening
events (See Figure ). Where once the signal died, the signal propagates to the end of the
domain by the opening of the pores in a process mediated by the diffusion of Ca2+ in the
gel. Furthermore, the traveling waves of depolarization and gel Ca2+ travel at speeds which
depend upon the dynamics of the pore. Thus, we see an elbow in the space time plots for
the wave: A fast wave that is diffusion dominant and an angled component, associated with
slower wave speed, when pore flickering sustains the wave.
We now explore the effects of different diffusion rates on the propagation speed of the
Ca2+ wave. We consider DCa to vary from √ 350µ2 /s down to 50µ2 /s. We expect that as
DCa decreases the wavespeed would scale as DCa .

2 2 2
DCa = 50μ /s DCa = 200μ /s DCa = 350μ /s

Figure 15: Plots of Ca2+ waves, similar to those in Figure (a,d) except we vary the diffusion constant from 50 up to 350µ2 /s.
We note that the wavespeeds slow with diffusion but not in a linear fashion. We calculate wavespeed by examining the time
the wave reaches 160µ and when it reaches 170µ. We find that the wavespeeds vary from c50 ≈ 1.25µ/s, c200 ≈ 1.852µ/s, and
c350 ≈ 2.222µ/s.

2.2

1.8
c

1.6

1.4

1.2
50 100 150 200 250 300 350
D Ca

√
Figure 16: A plot of wavespeed as a function of DCa . We note that the wavespeed scales as the DCa .

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 MBI Technical Report: work in progress

Buffering capability of the matrix

In this work, we have used parameters in order to obtain the qualitative behavior of the
observed dynamics. We have used a rapid buffering approximation for the proton dynamics
with a buffering coefficient of fHm . The value of fHm is not known and would likely vary in
different cell types as well as possibly vary in the life of the individual mitochondrion. We test
the role of the matrix buffering capability on the generation and propagation of Ca2+ and
potential traveling waves. The preparation is similar to the previous stimulation at time
t = 15 of a 5 micron strip about the origin with a consistent Ca2+ stimulus. As seen in Figure
, we vary the buffering capability of the mitochondria and find that proton buffering has a
profound effect on the generation of the traveling wave and its wavespeed. For preparations
with high buffering capability (that is fHm is small), the matrix effectively buffers transient
rises in pH due to the influx of Ca2+ into the cytosol and the sequence of pore opening events
fails to take place and the wave is never generated. As the proton buffering capability of the
matrix decreases (fHm rises), a traveling wave is generated and propagates. With further
decreases of the matrix pH buffering capability the resulting wavespeed increases. In Figure
, we see wave failure for strong pH buffering and the wave emerging for weaker pH buffering
with the wavespeed increasing linearly with fHm .

a) Ca C b) Ca C c) Ca C d) Ca C
300

200
t

100

0
0 100 200 0 100 200 0 100 200 0 100 200
x x x x

Figure 17: We vary the proton buffering coefficient from low to high. In a), the proton buffering capability is great
and fHm =2x10−7 . The traveling wave fails to terminate. In b), we decrease the proton buffering capability (that is, we
raise the proton buffering coefficient) so that fHm =2x10−6 . We note that a wave is generate and propagates at a wavespeed
approximately 1 µ/s. By increasing fHm further we note increased wavespeeds evident in c) and d), with fHm =1x10−6 and
fHm =2x10−5 , respectively. In all of these simulations, the calcium diffusion constant was set to DCa = 200µ2 /s.

High Conductance PTP Opening

The progression to an unhealthy state in vivo is likely a slow process where the level of
cytostolic calcium remains elevated for an extended period of time before a pore popping
event occurs. We propose a model that captures the qualitative behavior of the pore popping
event. Making the ansatz, that pore entering the high-conductance state to depends not
directly on mitochondrial calcium load, but rather a secondary slow process that depends
upon mitochondrial Ca2+ load, e.g., reactive oxidative species (ROS) or phosphate carriers.
In effect, mitochondrial Ca2+ must remain elevated for an extended period of time in order
to lead to a PTPh event.

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 MBI Technical Report: work in progress

Let P T Ph denote the gating variable of the pore opening to its high conductance state. The
dynamics are taken to be akin to a Hodgkin-Huxley-like gating variable

dP T Ph [(P T Pclosed)P T Ph∞(γ) − P T Ph (t)]

= , (11)
dt τh (CaM )

where P T Ph∞ is a function that depends upon a secondary process necessary for pore opening,
which we call γ. For simplicity, we take

P T Ph∞(γ) = Θ(γ − γ ∗ ) (12)

where Θ is the Heaviside function and γ ∗ is a threshold value that the secondary process
must to initiate opening to the high-conductance state. Similarly, we take the secondary
process, γ, to be governed by

dγ γ ∞ (CaM − Ca∗M ) − γ
= . (13)
dt τγ (CaM )

The constant Ca∗M is the threshold at which time the secondary process is activated. The
function γ ∞ could take on various forms: sigmoidal, Heaviside, or piecewise-linear. Taking
γ ∞ to be sigmoidal would mean that the time that calcium being above some threshold in
order to lead to pore popping would depend on the extent that the mitochondrial calcium
load is above the threshold. For simplicity, we consider γ ∞ to be the Heaviside function,Θ.
The key feature of this formulation is that past the threshold for mitochondrial calcium load,
Ca∗M , the secondary processes activates. In turn this slow secondary process must attain a
threshold γ ∗ whereby the pore activates and transitions to its high conductance state.
The time constants for these processes are taken to depend upon the mitochondrial calcium
load, whereby both changes in the secondary process and the transition to PTPh are slow for
low levels of CaM . In particular, we consider
!
6
5x10
τγ (CaM ) = 1000
+1 . (14)
cosh Ca M
1e−1

The time constant τγ is relatively large since this is to be the slower of the two processes
and take
τh (CaM ) = 100τγ (CaM ). (15)
In effect, once PTPh occurs, the PTP remains open because the mitochondrial calcium has
all been expunged into the cytosol.
Previously, we explored a stimulation protocol that induced PTPl - a reversible opening
that eventually results in the pore closing again. However, under longer slow subthreshold
Ca2+ stimulation, mitochondria can attain a breaking point whereby the pore pops and en-
ters into its open high-conductance state. Below, in Figure , are the results of a numerical
simulation of the pore transitioning to PTPh . A protocol for calcium stimulation that mim-
ics the slow degrade in Ca2+ sequestering in the cell is used. Calcium is infused a constant
rate that is sufficiently small so that the weak-acid flux can compensate and the pH remains

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 MBI Technical Report: work in progress

approximately at its original homeostatic value. The cytostolic calcium slowly rises, and the
mitochondrial calcium load in turn rises as it sequesters Ca2+ out of the cytosol. Eventually,
the mitochondrial load exceeds the threshold value of Ca∗M and the secondary process be-
comes activated. As Ca2+ infusion continues, the secondary process slowly rises, during this
time the mitochondrial Ca2+ load is elevated for an extended period of time. When the sec-
ondary process it attains its threshold vale γ ∗ , the PTP transitions to its high-conductance
state. The mitochondrial Ca2+ rushes out of the pore into the cytosol, where Ca2+ is weakly
buffered in comparison to the mitochondrial matrix. Thus the cytosolic Ca2+ is highly ele-
vated with no other mechanism for clearing Ca2+ , the cell would undergo Ca2+ cytotoxicity
whereby elevated cytosolic Ca2+ concentrations can activate cysteine proteases that mediate
plasma membrane breakdown through the cytoskeletal and plasma membrane proteins [12]
(in review by [6]).
The model compensates to preserve its diminished electrical potential by utilizing ATP to
eject H+ through the ATP-synthase. As protons are being ejected rather than admitted, the
mitochondrion enters a state where it relies solely on the TCA cycle for ATP production.
The cell cannot live in this state of limbo for an extended period of time, because the
mitochondrion is now an ineffectual a calcium store and calcium cytotoxicity will ensue.
Though we point out that, in this model, osmotic effects of the pore ’popping’ are neglected
and the mitochondria would likely swell and burst with the pore in its popped state. The
model of [22] tracks the K+ and Na+ fluxes which would be involved in swelling.

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 MBI Technical Report: work in progress

40 8

6
30
Ca*M
4
Ca C 20 Ca M
2
10

t2 0
t1
0
0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000
t t
7.7
0.8
7.65 γ* t2
0.6
7.6 t2
pH γ
7.55 0.4

7.5 0.2

7.45 t1
0
7.4
0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000
t t

160
140 t2 0.6
120
100
PTPH 0.4 t2
∆Ψ 80
60 0.2
40
20 0
0
0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000
t t

Figure 18: Calcium is continually infused into the cytosol at a sufficiently slow rate so that the pH remains at homeostasis.
In turn, the mitochondrial calcium load increases and eventually exceeds the threshold value of 4µm at time t1 ≈ 1800s,
activating the secondary process necessary for PTPh . Approximately 1700s later, the pore opens into PTPh at time t2 ≈ 3500.
The electric potential and the mitochondrial Ca2+ plummet at time t = t2 , and the cytostolic Ca2+ becomes incredibly elevated.

Discussion
As one reviews the modeling literature related to mitochondria and Ca2+ handling and the
supporting experimental work, several themes emerge. The first is that highly simplified
phenomenological models are unlikely to be sufficient as a starting point. This is because of
the complexity of mitochondrial physiology and the fact that coupling between the ER and
mitochondria appears to be significant. Second, we have to rely on modular formulations by
default. The myriad mechanisms involved have been characterized in isolation with mito-
chondria from different sources. Mitochondrial function almost certainly varies dramatically
between cell types, which is an unavoidable source of problems for interpretation. If we do

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 MBI Technical Report: work in progress

use a modular approach, then the parameter space is vast and the system is highly complex
and difficult to understand; in particular, because we are interested in nonlinear phenomena
like oscillations and waves which would be lost in linearized formulations. Therefore our
approach is to start with the complete and accepted modular model available (Magnus and
Keizer), to add the additional modules necessary for exploring wave propagation, and then
to reduce the model as far as we can in order to determine the necessary components.
At this stage, our model allows us to capture the novel behavior of the experimentally ob-
served mitochondria driven Ca2+ and potential waves in culture. Our work is a stepping
stone to understanding a myriad of issues pertaining to in vivo propagation of Ca2+ signals.
As mentioned previously, the study of Ca2+ dynamics has centered upon the trafficking of
Ca2+ between the ER and the cytosol. We aim to link our simplified model of mitochondria-
cytosol interaction to the Li-Rinzel ER-cytosol model [11] to study the interaction between
these two excitable media. We postulate that the mitochondria network will act in a manner
that will amplify or dampen calcium signals depending upon its strength. For simplicity
this could be done in point model then extended to the spatial case. Since the mitochondia
distributions vary depending upon cell type, we intend to explore the effects of hetero-
geneous spatial distributions of the mitochondrial network interacting with the ER. The
spatial interplay of the ER and the mitochondria will likely uncover traveling waves that
have non-constant wavespeeds, that is, it is a likely arena where a lurching wave exists.
While much remains elusive, it is clear is that the MPT is implicated in numerous diseases,
(e.g., Ca2+ dysregulation is implicated in human health problems including diabetes, cardiac
disease and Parkinson’s neurodegeneration [2]. This permeability transition is the nexus of
a large experimental effort principally because it appears that permanent activation of the
transition and subsequent depolarization of mitochondria are an essential initiating step
to most non-receptor mediated programmed cell death. At this point we have considered
a model for the pore-popping event whereby the pore opens to a high-conductance state
and remains open, ultimately leading to apoptosis or necrosis due to calcium cytotoxicity
or bursting due to osmotic effects. This model displays pore popping behavior under the
condition of high mitochondrial calcium load. The current model is phenomenological in
nature, as such a more detailed model that describes the pore forming process would be
of value. It would also be intriguing to study the roles of other factors associated with
MPT: reactive oxygen species, altered membrane lipids, electron transport chain anomalies
and altered gene expression. Computational modeling of mitochondrial Ca2+ dynamics may
therefore aid in the understanding of how to tip the balance of cell death towards acceleration
in the case of cancer therapies or towards protection in the case of neurodegenerative diseases.

Acknowledgments
Funding was provided by NSF DMS 0718558 and NIH MH-64611 to CPF, and NSF 0514356
to DT. The authors thank the Mathematical Biosciences Institute at The Ohio State Uni-
versity for hosting much of this project under NSF agreement 0112050. CPF is grateful for
the support of Joel Keizer and the discussions with him that informed much of this work.

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 MBI Technical Report: work in progress

Computational Methods

Point model calculations

The numerics for the point model were predominately carried out using the software package
XPPAUT developed by Bard Ermentrout.

Spatiotemporal caculations
We numerically simulate the system of partial differential equations with the method of lines
and an adaptive step algorithm using the software package MATLAB. The model contains
a wide range of scales making the problem numerically sensitive, that is, stiff. As such, the
spatial calculations are computationally expensive and time consuming.

Ca2+ Stimulation
In this work, we are principally concerned with two phenomena: the transient pore flickering
events and the permanent pore popping events. In order to induce that behavior we consider
two distinct Ca2+ stimulation protocols: (1) rapidly injecting pulses of Ca2+ to the cytosol
in order to evoke a flickering of the PTP and (2) a slow infusion of Ca2+ that eventually
leads to the pore popping – that is, opening to its high conductance state. This is meant
as an approximation of an ever increasing calcium load which could in part be due to a
decrease in the cells buffering capabilities (whether by the ER or the other mitochondria).
Mathematically, we represent the stimulation procedures (infusion and pulses) using the
following current:

N
X
Ca
Jstim = Ainf Θ(t − δinf ) + Apulse [Θ(t − tj ) − Θ(t − tj − δpulse )] (16)
j=1

where Θ represents the Heaviside function; that is, Θ(t) = 0 if t < 0 and Θ(t) = 1 if t > 0.
Note that a baseline level of calcium, Ainf , is infused for δinf seconds. Pulses of amplitude
Apulse and duration δpulse are applied at times tj .

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 MBI Technical Report: work in progress

Appendix
 
dCaC fC Ca Ca
= [M (Jna,ex − Juni − JPCaT P ) + Jstim
Ca
] (17)
dt Vc τmin
 
dCaM fM M Ca Ca
= [Juni − Jna,ex + JPCaT P ] (18)
dt Vm τmin
 
d∆Ψ M H
= [Jres − JFH0F 1 − JLH − JPHT P − JAN ADP Ca Ca
T − 2(Juni + JP T P )](19)
dt cmito τmin
 
dADPC 1 ADP ADP AT P
= [−MJAN T + C(Jhyd − Jgly )] (20)
dt VC γµ,m τmin
 
dADPM M ADP ADP ADP
= [JAN T − (JT CA + JF 0F 1 )] (21)
dt Vm γµ,m τmin
 
dNADHM M N ADH
= [Jred − JoN AD ] (22)
dt Vm γµ,m τmin

where τmin is a conversion factor so that time is in seconds and γµ,m is a conversion factor
so that the units are in µM. The constants fC correspond to the proportion of free Ca2+ in
the cytosol. The volume of the cytosol is denoted VC , and the total amount of protein in
the cytosol is given by C. The constants fM , VM , and M are similarly defined but for
the mitochondrial matrix. As mitochondria are effective calcium buffers, fM ≪ fC . The
meaning of the individual fluxes (the J terms) and where they can be found in the literature
is listed in the table below.

Fluxes
Notation Type Trafficked Source Equation(s)
Ca
Juni Uniporter Ca2+ Fall-Keizer 2001 [4] pg 158
Ca
Jna,ex Exchanger Ca2+ , Na2+ Magnus-Keizer 1997 [15] 21
Ca
Jstim Stimulation Ca2+ this work 16
JPCaT P PTP Ca2+ this work 2
H
Jres Respiration H+ Magnus-Keizer 1997 [15] 4,6
JFH0F 1 Through Synthase H+ Magnus-Keizer 1997 [15] 12, 14
JLH Leak H+ Magnus-Keizer 1997 [15] 7
JPHT P PTP H+ this work 1
ADP
JAN T Translocator ADP, ATP Magnus-Keizer 1997 [15] 16
ADP
Jhyd Hydrolysis ATP → ADP Magnus-Keizer 1998 [16] 35, 37
ADP
Jgly Glycolysis ADP → ATP Magnus-Keizer 1998 [16] 10
JTADP
CA Citric Acid Cycle ADP → ATP Magnus-Keizer 1998 [16] 24
ADP
JF 0F 1 ATP-synthase ADP → ATP Magnus-Keizer 1997 [15] 4, 6
N ADH
Jred Reduction NAD → NADH Magnus-Keizer 1997 [16] 21
JoN AD Oxidation NADH → NAD Magnus-Keizer 1997 [15] 4, 5

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 MBI Technical Report: work in progress

Compartmental equations
M = V pmito dmito
protein amts (mg)
C = V pcytol dcyto
VM = V pmito
compartmental volumes (ml)
VC = V pcyto
− log10 H106 pH from H+


pH =
H = 10(−pH) 106

Parameters and ICs for Channel Formulation

p1 = 0.0216 CaC (0) = 0.419457395
p2 = 10−9 CaM (0) = 2.49110278
p3 = 0.0231 Ψ(0) = 166.4550752
p4 = 0.0001 HM (0) = 0.02403069
ampτ = 104 P T Pℓ (0) = 2.215121963756248e − 05
p6 = 0.01 ADPM (0) = 4.23359810689
fHM = 1.28x10 −7 ADPC (0) = 0.01558366827782595
κHM = 3500 N ADHM (0) = 2.4753974569
κCa = 400
fC = 0.01
fM = 0.0003
betweenCAnoF licker = 44 betweenCAF licker = 7
AmpCa = 6200 glc = 3

Parameters used for spatial simulations

p1 = 0.023 CaC (0) = 0.4
p2 = 10 −9 CaM (0) = 2.7
p3 = 0.023125 Ψ(0) = 162
p4 = 10 −9 HM (0) = 0.02403
ampτ = 106 P T Pℓ (0) = 0
p6 = 0.1 ADPM (0) = 7.6
fHM = 1.8x10−6 ADPC (0) = 0.01
κHM = 60 N ADHM (0) = 0.16
κCa = 60
fC = 0.01 glc = 1
fM = 0.0003
AmpCa = 4000

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 MBI Technical Report: work in progress

Nucleotide conservation
8 dmito
NAD N AD = µ − N ADHM
12 dmito
mitochondrial ATP AT PM = µ − ADPM
cytostolic ATP AT PC = 2 − ADPC
mitochondrial free ATP ADPM,f ree = 0.8 ADPM
cytostolic free ATP ADPC,f ree = 0.3 ADPC
3−
charged nucleotides ADP(C,M ) = 0.45 ADP(C,M ),f ree
charged nucleotides M gADPC− = 0.55 ADPC,f ree
4−
charged nucleotides AT P(C,M ) = 0.05AT P(C,M )

29
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