From the Mathematical Research Branch, National Institutes of Health, Bethesda, Maryland; Department of Physiology and § Department of Mathematics, University of Michigan, Ann Arbor, Michigan 48109-1109
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ABSTRACT |
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The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10-12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675-2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81-84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.
Key words: inositol 1,4,5-trisphosphate receptor; phosphorylation; protein kinase A; mathematical model; calcium oscillations ![]() |
INTRODUCTION |
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The production of the intracellular signaling factor inositol 1,4,5-trisphosphate (IP3)1 and the subsequent release
of Ca2+ stored in intracellular organelles is a fundamental cellular signaling function (Berridge, 1997). The form
of the resultant change in the intracellular free Ca2+ concentration ([Ca2+]i) is highly variable. Specific, well-characterized response types include a single, large increase
in [Ca2+]i, which may virtually deplete the stores, and
smaller, maintained oscillations of [Ca2+]i (Berridge,
1990
). Often both patterns may be observed in a single
cell type, depending on the agonist concentration.
The release of Ca2+ by IP3 occurs through the activation of a specific receptor for IP3, which is located on
the endoplasmic reticulum (ER) surface, and which is
also a functional calcium channel (Bezprozvanny and
Ehrlich, 1995; Taylor and Traynor, 1995
; Joseph, 1996
;
Yoshida and Imai, 1997
; Taylor, 1998
). In addition to its
IP3 binding site (at the NH2-terminal end) and its pore-forming region (the COOH-terminal end, containing
six membrane-spanning regions), the IP3 receptor (IP3R)
also contains a large regulatory domain between the
NH2- and COOH-terminal regions. A number of cytosolic factors have been suggested to modulate IP3R
activity, including, but not restricted to, PKA, PKC,
Ca2+-calmodulin kinase II (CaMK-II), adenine, and
possibly guanine nucleotides. These additional factors
may permit the wide range of response types that are
seen for IP3-induced Ca2+ release.
In addition to factors such as kinases modulating
IP3R activity, Ca2+ itself plays a fundamental role. A
number of studies have shown that the steady state
open probability of the type-I IP3R displays a bell-shaped dependence on [Ca2+]i, with a peak at approximately [Ca2+]i = 300 nM (Bezprozvanny and Ehrlich,
1995; Taylor and Traynor, 1995
; Joseph, 1996
), while
for type-III IP3 receptors the open probability curve is a
monotonically increasing function of [Ca2+]i (Hagar
et al., 1998
). However, the steady state behavior of the
receptor is much less important than the dynamic response of the receptor to a sudden increase of either
[Ca2+]i or [IP3]. When exposed to a sudden change in
[Ca2+]i or [IP3], the receptor responds by a transient
increase in open probability, followed by adaptation or
recovery, to a lower level (Finch et al., 1991
; Dufour
et al., 1997
; Marchant and Taylor, 1998
). Direct injection of IP3, or the use of nonhydrolyzable IP3 analogues, has shown that in mouse pancreatic acinar cells oscillations of [Ca2+]i can occur when the IP3 concentration is constant (Wakui et al., 1989
; Berridge and
Potter, 1990
). Thus, dynamic regulation of the receptor
by Ca2+ may represent the mechanism by which oscillations of [Ca2+]i arise, with Ca2+ initially promoting its
own release, and then secondarily inhibiting further release. In the DISCUSSION, we discuss the possible effects on the model of changing IP3 concentrations.
A number of mathematical models have been developed to simulate IP3-induced Ca2+ release (De Young
and Keizer, 1992; Atri et al., 1993
; Sneyd et al., 1995
;
Tang et al. 1996
), although, to date, all models have
been based on the properties of the type-I receptor. In
the model of De Young and Keizer (1992)
, each subunit of the tetrameric IP3R contains one binding site
for IP3 and two binding sites for Ca2+, an activating site
and an inactivating state. It was assumed that Ca2+ flux
occurred only when the IP3 binding site and the activating Ca2+ binding site were occupied, and the Ca2+ inactivating site was unoccupied. This formulation resulted
in an eight-state model, only one of which was a conducting state. With rate constants for interconversion
between the eight states chosen, where possible, to correspond to experimental values, this model displayed
oscillatory behavior over a range of [IP3] (De Young
and Keizer, 1992
). A fundamental feature of this
model, necessary to allow oscillations to occur, is that
IP3 and Ca2+ binding to the activation site are fast processes, whereas Ca2+ binding to the inhibitory site is a
slow process. Other models of IP3R kinetics (Atri et al.,
1993
; Sneyd et al., 1995
) are based on the same premise
of fast activation and slow inhibition by Ca2+, and Tang
et al. (1996)
showed that these are essentially equivalent formulations. A model of [Ca2+]i dynamics in pituitary
gonadotroph cells (Li et al., 1994
, 1997
) is perhaps the
most highly developed of these in terms of the specific
cellular mechanisms controlling [Ca2+]i and equivalence
of the model behavior to experimental single cell data.
Experimental data on the relative rates of activation
and inactivation of the IP3R by Ca2+ is consistent with
inactivation being a slower process (Parker and Ivorra,
1990). However, careful analysis of data with higher kinetic resolution (Finch et al., 1991
; Dufour et al., 1997
;
Marchant et al., 1997
; Marchant and Taylor, 1998
) suggests that the difference between the rates of activation
and inactivation is not as great as is typically used in
mathematical models. Furthermore, despite the fact
that inactivation is apparently too slow in the models, the models are unable to reproduce long-period, baseline oscillations (up to 2 min or longer in period) observed in cells such as hepatocytes (Kawanishi et al.,
1989
) and pancreatic acinar cells (Yule et al., 1991
). Recent models have addressed the issue of long-period oscillations (Dupont and Swillens, 1996
; Laurent and
Claret, 1997
), but their relevance to any particular cell
type is, as yet, unclear.
From the point of view of modeling Ca2+ oscillations
and waves, pancreatic acinar cells present particular
difficulties. Firstly, in response to different agonists,
pancreatic cells exhibit markedly different Ca2+ responses (Petersen et al., 1991b; Yule et al., 1991; Lawrie et al., 1993
; Thorn et al., 1993b
). Application of acetylcholine (ACh) to pancreatic acinar cells results in the
generation of [Ca2+]i oscillations that are roughly sinusoidal with a frequency of ~4-6/min. The [Ca2+]i oscillations are superimposed on a raised baseline such that [Ca2+]i does not fall to basal levels before the generation
of the next peak (Yule et al., 1991
). In contrast, the
[Ca2+]i response to cholecystokinin (CCK) is very different, consisting of baseline spikes of much longer period.
Despite these differences, there is much evidence
that both types of oscillations result from agonist-
dependent activation of phospholipase C, and the resulting increase in intracellular [IP3] (see DISCUSSION).
How might these two very different patterns of Ca2+ release occur, with apparently the same basic intracellular
processes? In particular, how do the long baseline
spikes observed with CCK occur, given that [IP3] is
thought to be maintained above basal levels throughout the period of stimulation, whereas [Ca2+]i, which
initially provides the negative feedback signal to end Ca2+ flux from the stores, has returned to its basal values long before the next spike occurs? In studying
these questions, it is important to keep in mind that
pancreatic acinar cells contain a preponderance of
type-III IP3 receptors (Wojcikiewicz, 1995), and thus
any model to explain the observed oscillations should
be based on data from the correct receptor subtype
wherever possible. Fortunately, data on the steady state
properties of type-III IP3 receptors have recently become
available, although their adaptational and time-dependent properties are still unknown in detail.
Our goal is to understand the mechanisms underlying these different oscillatory patterns and provide a
single explanation for these seemingly diverse phenomena. Of course, such an explanation can be simplistic at
best and cannot take into account all the complexities
of the real cell. Nevertheless, it will provide a framework to help us understand how a set of relatively simple cellular interactions can result in diverse and complex behavior. We begin by showing (experimentally)
that in mouse and rat pancreatic acinar cells physiological concentrations of CCK cause rapid phosphorylation of the type-III IP3R, but physiological concentrations of ACh do not. Assuming that the phosphorylated
receptor does not pass Ca2+ current (see DISCUSSION),
it follows that the rate at which the IP3R recovers from
inactivation depends crucially on the agonist. Secondly,
we show that the rate of Ca2+ removal from the cytoplasm is an order to magnitude slower after application
of CCK than after application of ACh. These data are then incorporated into a model of the type-III IP3R,
and thus into a whole-cell model of the Ca2+ response.
The model provides a unified explanation of seemingly disparate results; it agrees with the rates of activation
and inactivation of the IP3R by Ca2+, as measured by
Finch et al. (1991) and Dufour et al. (1997)
, but can
nevertheless produce oscillations of very long period and generate oscillations of the two observed types.
Furthermore, the steady state open probability of the
model type-III IP3 receptor is an increasing function of
Ca2+, as found experimentally by Hagar et al. (1998)
,
thus showing that such monotonic steady state curves
are consistent with oscillatory behavior, a fact that is not
always appreciated. Predictions of the model are then
tested, and confirmed, experimentally.
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METHODS |
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Materials
Fura-2/AM was purchased from Molecular Probes, Inc., collagenase (CLSPA grade) from Worthington Biochemicals, bovine serum albumin (fraction V) from ICN Immunobiologicals, and minimal essential amino acid supplement from GIBCO BRL. [32P]orthophosphate (9,000 Ci/mmol) was obtained from Dupont NEN. Monoclonal antisera directed against the type-III inositol 1,4,5-trisphosphate receptor was obtained from Transduction Laboratories. All other materials were obtained from Sigma Chemical Co.
Preparation of Pancreatic Acini
Acini were prepared by methods previously described (Williams
et al., 1978; Yule et al., 1996
). In brief, pancreata were excised from freely fed adult male Sprague-Dawley rats (200-250 g) or mice. Acini were prepared by enzymatic digestion with purified collagenase, followed by mechanical shearing. Acini were then filtered through 150 µm Nitex mesh, purified by sedimentation through 4% BSA in HEPES ringer, and then suspended in a physiological salt solution containing 10 mg/ml bovine serum albumin, 0.1 mg/ml soybean trypsin inhibitor, and (mM): 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1.0 Na2HPO4, 10 HEPES,
2 L-glutamine, 5.5 D-glucose, essential amino acids. The pH was
adjusted to 7.4 and equilibrated with 100% O2.
Measurement of [Ca2+]i
Isolated acini were incubated with 2.5 µM fura-2/AM at ambient
temperature for 30 min, and then washed and resuspended in fresh physiological salt solution without BSA. For measurement of [Ca2+]i, fura-2-loaded acini were transferred to a chamber,
mounted on the stage of an Axiovert 35 microscope (Carl Zeiss,
Inc.), and continuously superfused at 1 ml/min with PSS without
BSA. Solution changes were rapidly accomplished by means of a
valve attached to an eight-chambered superfusion reservoir,
which was maintained at 37°C. Determination of [Ca2+]i was performed using digital imaging microscopy with an ATTOFLUOR ratiovision system (ATTO Inc.) as previously described (Yule et al., 1996). Briefly, excitation at 340/380 nm was alternately achieved by a computer controlled filter and shutter system and the resultant emission at 505 nm was recorded at the rates indicated in the figures by an intensified CCD camera, and subsequently digitized. Mean gray values obtained by excitation at 340 and 380 nm, in user-defined areas of interest, were used to compute 340/380 ratios. Calibration of fluorescent ratio signals was
accomplished as previously described according to the equation
of Grynkiewicz et al. (1985)
by comparing the fluorescence of
known standard Ca2+ buffers containing fura-2.
Assessement of Phosphorylation of the Type-III IP3 Receptor
Acini were labeled with 0.3 mCi/ml 32PO4 for 2 h in HEPES ringer devoid of added phosphate. Aliquots of labeled acinar cells were then treated as indicated. At the end of the incubations, the acini were rapidly centrifuged in a microcentrifuge and the pellets were resuspended in ice-cold lysis buffer containing 100 mM NaF, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM benzamidine, 1% Triton X-100, 10 µg/ml leupeptin, and 10 µg/ml pepstatin, at pH 7.4). The homogenates were then sonicated. After 30 min on ice, the samples were precleared by addition of 100 µl of protein A-agarose beads and rotation for 2 h at 4°C. The protein A beads were removed by centrifugation and the protein content of the samples was assayed. Type-III IP3R was immunoprecipitated from the samples of equal protein by incubation with 1 µg antibody/mg of protein (Transduction Laboratories) for 3 h. After incubation, the beads were washed five times with the lysis buffer by centrifugation and resuspension. After the final wash, the beads were boiled in Laemmli buffer and immunoprecipitated proteins separated by electrophoresis on 5% SDS-PAGE gels. The gel was dried and exposed to a Phosphoimager intensifing screen (Bio-Rad Laboratories) for visualization and analyzed by molecular analyst software for quantification (Bio-Rad Laboratories).
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RESULTS |
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Secretagogue phosphorylation of the type-III IP3 receptor.
Aliquots of acini (in duplicate), metabolically labeled with
32P, were stimulated with secretagogues for 2 min and
the extent of phosphorylation of the receptor was assessed by immunoprecipitating the receptor, separation on SDS-PAGE, and then autoradiography. Acini
were initially treated with varying concentrations of
CCK. In unstimulated acini, phosphorylation of one major band of ~300 kD, corresponding to the type-III IP3R,
could be detected. An increase in the degree of phosphorylation could be demonstrated at 10 pM CCK (170 ± 15% above basal) and reached a maximum at 100 nM
CCK (252 ± 23% of control). Phosphorylation of the receptor was not significantly greater at 1 or 10 nM CCK
(n = 4 rat and 2 mouse preparations gave qualitatively
similar results). It should be noted that the onset and
maximum phosphorylation of the receptor achieved coincides with concentrations of CCK that can be demonstrated to induce calcium oscillations (Yule et al., 1991,
1993
). Fig. 1 shows a typical experiment. The extent of
receptor phosphorylation was also investigated upon
stimulation by the muscarinic agonist, carbachol. In this
series of experiments, an increased phosphorylation of
the type-III IP3R was also consistently observed 2 min after stimulation with agonist. Phosphorylation could be
detected at 1 µM CCh (160 ± 22%) and reached a peak
at 10 µM (203 ± 28% of control). In contrast to stimulation by CCK, no significant phosphorylation of the receptor was observed at concentrations of CCh below 1 µM,
concentrations that can be demonstrated to induce an
oscillatory calcium signal (Yule et al., 1991
; n = 3 rat and
2 mouse preparations gave quantitatively similar results).
A typical experiment is shown in Fig. 2.
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Phosphorylation of the type-III IP3 receptor by second messengers. To investigate the mediator generated upon agonist stimulation that results in phosphorylation of the type-III IP3R, duplicate aliquots of acini were incubated for 5 min with agents known to activate or be a mediator in discrete second-messenger pathways: acini were incubated in either cyclopiazonic acid (CPA), which leads to an elevation of [Ca2+]i by inhibition of the Ca2+-ATPase present on intracellular calcium stores, TPA, an activator of protein kinase C, or CPT-cAMP, a cell-permeable cAMP analog. In three experiments (n = 2 rat and 1 mouse preparation) no enhanced phosphorylation of the type-III IP3R was ever observed from acini incubated with either TPA or CPA, indicating that a calcium-dependent kinase or protein kinase C is apparently not responsible for phosphorylation of the receptor. A marked increase in phosphorylation was, however, always observed when the acini were incubated with CPT-cAMP. The extent of phosphorylation, >427 ± 52% above basal, was greater than observed with phospholipase C-coupled agonists. These data indicate that a cAMP-dependent pathway is capable of phosphorylating the receptor. A typical experiment is shown in Fig. 3.
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Model Construction
Previous mathematical models of the IP3R have tended
to treat IP3 as a permissive factor; its binding is regarded as obligatory for channel activation, but beyond
that it has a passive role, with [Ca2+]i providing the dynamics underlying oscillations. However, two lines of
experimental evidence suggest that IP3 binding may
play a more important, dynamic role. Firstly, binding
studies have shown the affinity of IP3 for its receptor is
dependent on the [Ca2+]i. For type-I receptors, increasing [Ca2+]i causes either a decrease in IP3-binding affinity (Yoneshima et al., 1997) or a biphasic effect (Sienaert et al., 1997
), whereas, for the type-III receptor, increasing [Ca2+]i enhances IP3 binding (Yoneshima et al.,
1997
). Secondly, there is evidence that IP3 itself may inactivate the IP3R. For example, experiments with high
kinetic resolution performed under Ca2+-clamp conditions have shown that during continuous perfusion with a particular medium-Ca2+ concentration, the introduction of IP3 causes a transient release of 45Ca2+
from microsomes derived from rat brain synaptosomes
(Finch et al., 1991
) or rat hepatocytes (Dufour et al.,
1997
; Marchant and Taylor, 1998
). This suggests either
that IP3 can inactivate the receptor or that at least some
of the Ca2+-induced inactivation occurs only during
permeation of the channel. The Mn2+ quench experiments of Hajnóczky et al. (1993)
demonstrate that IP3
can inactivate the receptors without permeation of
Ca2+. Furthermore, they found that Ca2+ enhanced IP3-induced channel inactivation.
Reaction scheme.
We assume that the complete IP3R is
composed of four, functionally identical, independent
subunits. The reaction scheme governing transitions of
each subunit is shown in Fig. 4. S denotes the fraction
of subunits in the shut state (S), in which the receptor channel is closed and IP3 is not bound. Binding of IP3
causes the receptor to be converted to the open state,
O, and we let O (Fig. 5) denote the fraction of receptors in state O. Although several subconductance states
of IP3R have been observed, the channels most often
open to just one of these (Watras et al., 1991). We
therefore assume that IP3 must be bound to all four subunits for the receptor to be in the conducting state.
Thus, the fraction of conducting receptors is O4.
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Receptor kinetics. By the law of mass action differential, equations for the various receptor states can be determined. The equation for the fraction of receptor subunits in the open state (O) is
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Simulation of IP3- and Ca2+-clamp experiments.
To determine reasonable values for the transition rates, we simulated experiments in which a superfusion system was
used to determine the rapid kinetics of IP3R activation
and inactivation. This technique has the feature that
extravesicular [IP3] and [Ca2+] can be rapidly changed
under otherwise clamped conditions (Finch et al.,
1991; Dufour et al., 1997
; Marchant and Taylor, 1998
). Unfortunately, such data are not available from type-III
receptors, and so it is unknown whether their time-
dependent behavior is similar to that of type-I or -II receptors. In the absence of evidence to the contrary, we
shall assume it is, although the model does point out
some likely important differences, as we shall see.
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Monotonic steady state [Ca2+] dependence.
For type-I IP3
receptors, the steady state open probability has been
found to have a bell-shaped dependence on [Ca2+]
(Bezprozvanny and Ehrlich, 1995; Taylor and Traynor,
1995
; Kaftan et al., 1997
), and this can easily be reproduced by our model (computations not shown). However, recent results (Hagar et al., 1998
) have shown that
the type-III IP3R has a steady state open probability
curve that is a monotonic increasing function of Ca2+.
Since it appears that Ca2+ oscillations in pancreatic acinar cells are governed by Ca2+ release through type-III
IP3R, we chose parameter values so as to obtain a
monotonically increasing steady state open probability curve as a function of Ca2+ concentration (Fig. 7).
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Incorporation into a whole-cell model. We now incorporate the receptor model into a description of acinar cell [Ca2+]i responses, endeavoring to keep the model as simple as is reasonably possible so as to retain the focus on the kinetics of the IP3R. Hence, we do not include any possible effects of ryanodine receptors (see DISCUSSION).
The equation for [Ca2+]i (c) is given by
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Theoretical Results
Parameter values.
The parameter values used in the
model simulations are presented in Table I. The values
for the rates of activation and inactivation of the IP3 receptor by Ca2+ (1,
1,
2,
2,
2,
3,
3, k
1, and k2)
were determined by ensuring the model agrees with
the results of Dufour et al. (1997)
. This was not done by
a detailed fitting procedure, only approximate agreement was required. The parameters governing the rate
of phosphorylation of the IP3 receptor by PKA (
4 and
4), and the recovery from the I2 state (k5), were not determined from experimental data; the values in Table I
were chosen so as to give reasonable agreement with
the observed CCK-induced oscillations. For ACh-induced
oscillations,
4 was set to zero to mimic the much decreased rate of receptor phosphorylation.
Pumping rates. To measure the approximate rate of Ca2+ ATPase activity, exponential functions were fit to the downward slopes of the oscillations induced by either ACh or CCK (data not shown). Although this assumes that the decline in Ca2+ concentration during the decreasing phase of the oscillation is due to Ca2+ pumping, and although it assumes the pumping has linear kinetics, these assumptions will have little, if any, qualitative effect on the result, and are accurate enough for our purposes; we are not doing a detailed fit of the model to data, merely using the data to determine approximate values for the parameters.
ACh-induced oscillations have a declining phase that approximates an exponential curve, with a decay rate that varies from ~0.2 to ~0.4 sCalcium influx.
There remains only one parameter,
the rate of Ca2+ influx, to discuss. We hypothesise that
this parameter is agonist dependent. In addition to the
very different kinetics of [Ca2+]i transients evoked by the
two agonists, the transients also differ greatly in their
sensitivity to removal of external Ca2+. ACh-induced
transients are very sensitive, such that if the medium
bathing cells is switched from normal (1.2 mM [Ca2+])
to nominally Ca2+-free during repetitive [Ca2+]i oscillations, the oscillations are abolished within ~30 s (Yule et al. 1991). This behavior has also been observed by
Muallem et al. (1990)
, and for bombesin-stimulated oscillations in rat pancreatic acinar cells by Xu et al.
(1997)
.
ACh-induced oscillations.
We investigated the effects of
increasing [IP3] on [Ca2+]i responses in the model,
with the pathway regulated by k4 switched off. The rates
of Ca2+ influx and pumping were set to 0.4 and 2.6 µM
s1, respectively (see Table I). Raising [IP3] to 0.66 µM
caused the generation of [Ca2+]i oscillations with a period of ~15 s (Fig. 8). During the oscillatory behavior,
the nadir of the oscillations occurred at ~300 nM, well above the resting [Ca2+]i.
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CCK-induced [Ca2+]i oscillations.
The experimental results indicate that CCK (but not ACh) activates PKA,
which phosphorylates the inactivated (I1 state) receptors, converting them to the I2 state. The receptors remain in the I2 state, where they are insensitive to
[Ca2+]i, until the action of phosphatases converts them
back to the S state. The action of PKA was modeled by
increasing k4 to 0.05 s1. Furthermore, we reduced the
rate of Ca2+ turnover by decreasing Vp, the maximum
pump rate, to 0.2 µM s
1, while the rate of Ca2+ influx
was reduced to 0.025 µM s
1.
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Testing Model Predictions
Experiments with Ca2+-free media.
We used the model to
investigate the relationship between the rate of Ca2+ influx and generation of ACh-induced [Ca2+]i oscillations. By reducing Jinflux, we can simulate the effect of
reducing extracellular [Ca2+]. It turns out that, as Jinflux
is decreased, the range of IP3 concentrations for which
oscillations are observed shifts to the right; i.e., to
higher [IP3]. Thus, a decrease in calcium influx would
be expected to abolish oscillations, while an increase in [IP3] would be expected to restore them. This behavior
is demonstrated in Fig. 12 A. The values for Jinflux and
[IP3] are indicated at the top of the figure. At the
normal Jinflux value of 0.4 µM s1, oscillations are induced when the concentration of IP3 is raised to 0.66 µM. Reducing Jinflux to 0.35 µM s
1 abolishes the oscillations, but these are recovered when [IP3] is increased
to 0.75 µM.
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Effects of phosphorylation on ACh-induced oscillations.
Using the model, we studied the effects of phosphorylating the IP3 receptors during ACh-induced oscillations (computations not shown). Oscillations are initiated by 0.66 µM IP3, with the other parameters the
same as for ACh-induced oscillations (Table I); in particular, 4 = 0 to simulate the absence of phosphorylation. Then, at 100 s,
4 was increased to 0.05 s
1, to simulate the cAMP-induced phosphorylation of the receptor by PKA. Oscillations are quickly abolished. This
prediction is confirmed by existing experimental data
(Camello et al., 1996) and our own experiments, in
which oscillations induced by 100 ACh are quickly abolished by the addition of 0.1 mM CPT-cAMP (Yule, D.I.,
unpublished data).
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DISCUSSION |
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Based on old and new experimental data, we have constructed a new model of the type-III IP3R and incorporated it into a whole-cell model for intracellular Ca2+
oscillations in pancreatic acinar cells. This model
agrees with recent data on the rates of activation and
inactivation of the IP3R by Ca2+ (Dufour et al., 1997),
and can reproduce both the short-period, raised baseline oscillations induced by ACh in pancreatic acinar cells, as well as the long-period baseline spiking induced by CCK. The steady state open probability of the
model IP3R is a monotonically increasing function of
[Ca2+]i, as shown experimentally by Hagar et al.
(1998)
.
The ability of the model to reproduce this wide variety of experimental data is based principally on two
things. (a) Physiological concentrations of CCK cause
rapid phosphorylation of the IP3R while physiological
concentrations of ACh do not. Phosphorylation of the
IP3R does not appear to be Ca2+ dependent, and can
also result from the addition of CPT-cAMP. Hence, we
hypothesize that CCK, via production of cAMP and activation of PKA, shunts the IP3R through a phosphorylation pathway that prevents rapid recovery from receptor inactivation. Inactivation of the IP3R by Ca2+ is
rapid, as indicated by the data of Dufour et al. (1997),
and then receptor phosphorylation holds the receptor
in an inactivated state. The resulting long-period oscillations are then governed by the time taken to recover
from the phosphorylated state. In the absence of phosphorylation (i.e., after stimulation by ACh), the oscillation period is governed principally by the rate of recovery from Ca2+-induced inactivation of the receptor. (b)
In the presence of ACh, the flux of Ca2+ in both directions across the plasma membrane is greater than in
the presence of CCK. Measurement of the rates of Ca2+
removal during the downstroke of the oscillations indicates that the rate of Ca2+ ATPase activity is much
greater in the presence of ACh than in the presence of
CCK. Furthermore, it appears that ACh increases the
rate of Ca2+ entry from outside the cell much more
than does CCK. Indirect evidence for this has appeared
before; Yule et al. (1991)
showed that removal of external Ca2+ abolishes ACh-induced oscillations, but has little effect on CCK-induced oscillations. By making this
assumption in the model, we were able to predict the
effect of both reducing external Ca2+ and increasing
agonist concentration. The fact that the model predictions were confirmed by the experimental data lends
support to this hypothesis, although this evidence is
still indirect.
It has been known for some years that PKA is able to
phosphorylate the IP3R; more recently, Wojcikiewicz
and Luo (1998) have shown that type-I, -II, and -III receptors are differentially susceptible to phosphorylation in intact cell lines (AR4-2J rat pancreatoma and
RINm5F rat insulinoma cells). Furthermore, it is also
known that CCK activates both the adenylate cyclase
and phospholipase C pathways in pancreatic acinar
cells, while ACh does not appear to activate the adenylate cyclase pathway (Schulz, 1989
; Petersen and Wakui,
1990
). However, what is far less clear is the exact effect
of IP3R phosphorylation. In some cell types, it appears
that phosphorylation of the IP3R by PKA inhibits Ca2+
release (Supattapone et al., 1998; Volpe and Alderson-Lang, 1990
), while in hepatocytes the opposite effect
occurs (Hajnóczky et al., 1993
; Joseph and Ryan, 1993
).
In platelets, PKA phosphorylation of the IP3R causes a
30% inhibition of IP3-induced Ca2+ release (Quinton
and Dean, 1992
), while more recent data shows that
PKA inhibits IP3-induced Ca2+ release in megakaryocytes (Tertyshnikova and Fein, 1998
). In a renal epithelial cell line, kinase activators and phosphatase inhibitors decrease the response to carbachol, while kinase
inhibitors increase the response to carbachol (Xu et al.,
1996a
), results that are consistent with the assumptions
of our model.
Our experiments on the phosphorylation of the IP3
receptor were performed in both rats and mice, and
similar results were obtained. Thus it is reasonable to
assume that the model of the IP3 receptor is applicable
to both rats and mice. However, there are quantitative
differences between the Ca2+ responses of rat and mice
pancreatic acinar cells to agonist stimulation. In the
study of Tsunoda et al. (1990) in rat pancreatic acinar
cells in intact acini, Ca2+ responses to application of
CCh took the form of low period baseline spiking, similar to the responses to application of CCK. Since most
of the studies showing clear differences between the responses to CCK and ACh have been performed in single isolated mouse pancreatic acinar cells (Osipchuk
et al., 1990
; Yule et al., 1991
; Petersen et al., 1991a
; Lawrie et al., 1993
; Thorn et al., 1993b
), it is not clear
whether our conclusions are also applicable to rat pancreatic acinar cells in an acinus. For a start, it is known
that gap junctional communication modulates the observed Ca2+ oscillations in rat pancreatic acini (Stauffer
et al., 1993
), but whether this is sufficient to account
quantitatively (or even qualitatively) for the differences
in the responses of coupled and isolated cells is not
known. Furthermore, it is likely that variations in crucial parameter values, such as pumping and influx rates, will lead to quantitatively different model behavior, and such differences may be one way to explain the
differing responses of rat and mice pancreatic acinar
cells. However, we have not done exhaustive parameter
studies and so cannot say for certain. Note, however,
that baseline spiking in response to application of ACh
is also seen in mouse pancreatic acinar cells, as long as
the concentration of ACh is low enough. Hence, our
model is consistent with the data from rat pancreatic
acinar cells in this low concentration limit. It is thus
plausible that the basic mechanisms are similar in both
rat and mice pancreatic acinar cells, differing only in
the details of certain parameter values.
In our model, we have assumed that phosphorylation of the IP3R shunts it into a closed state; it is important to note that this is assuming that phosphorylation neither inhibits nor potentiates Ca2+ release. Hence, the assumptions underlying our model are consistent with both effects of PKA. This can easily be seen by the following argument. If k3 were small, then the intrinsic recovery of the IP3R from the inactivated state would be slow; if the rates of phosphorylation and dephosphorylation (k4 and k5) were large enough, it is possible that shunting the receptor through the phosphorylated I2 state could increase the steady state open probability. Conversely, if k3 were large, and thus the intrinsic recovery from inactivation was fast, then shunting the receptor through the phosphorylated state could decrease the steady state open probability. In fact, we can derive an explicit relationship between k3, k4, and k5 that will determine the exact effects of phosphorylation on the steady state open probability. The steady state proportion of open receptors, O, can be easily calculated as
![]() |
(9) |
![]() |
(10) |
Thus, the function controls the sensitivity of the IP3R
to IP3; as
increases, the sensitivity of the IP3R decreases, and vice versa. A short calculation now shows
that
is an increasing function of k4 when 2k3 > k5
k4, and is a decreasing function of k4 when 2k3 < k5
k4. It follows that, depending on the values chosen for
k3, k4, and k5, the model can reproduce either an increase or a decrease of sensitivity of the IP3R upon phosphorylation.
Oscillations in the model occur for constant [IP3],
and are the result of cycles of activation and inactivation of the IP3R. At present, there is no direct evidence
for our assumption that oscillations in [IP3] are not
necessary to obtain [Ca2+]i oscillations. From experiments using nonmetabolizable analogs of IP3, there is
indirect evidence that the observed [Ca2+]i oscillations
are not governed by underlying oscillations in [IP3]
(Wakui et al., 1989; Thorn et al., 1993b
). However, a
complete resolution of this question awaits an experimental determination of the kinetic behavior of [IP3]
during the course of a [Ca2+]i oscillation. During application of an agonist, [IP3] will certainly rise and fall as
IP3 is produced and degraded. We do not build these
features into our model directly, but just assume that
IP3 is produced and degraded with given rates. Thus, in
the simulations, [IP3] increases and decreases gradually. This has little effect on the oscillatory behavior of
the model, which can be well understood by considering the behavior at fixed [IP3].
Although it appears that ryanodine receptors do exist
in pancreatic acinar cells, we have not included in the
model any possible effects of ryanodine receptors or
IP3-independent Ca2+-induced Ca2+ release. Kasai et al.
(1993) have shown that the response to ACh is eliminated by heparin, although there is still Ca2+-induced
Ca2+ release from the granular area. Thus, although it
is likely that IP3-independent factors are important for
modulating the shape of the [Ca2+]i oscillations, it appears that they cannot, by themselves, support oscillations. Nathanson et al. (1992)
have also shown that
Ca2+-induced Ca2+ release has an effect on the speed of
ACh and CCK-stimulated intracellular [Ca2+]i waves.
However, neither caffeine nor ryanodine eliminates the waves, although they do decrease the wave speed. Furthermore, the intracellular waves are initiated at the
apical zone of the cell, which is where the type-III IP3
receptors are mostly found (Nathanson et al., 1994
).
We interpret these results to mean that the oscillatory
period and other fundamental oscillation properties are determined primarily by the properties of the IP3
receptors in the apical zone, with other forms of Ca2+-induced Ca2+ release playing only a modulatory role.
Hence, for simplicity, in this model we consider only
IP3 receptors.
One major difference between our model and previous ones is in the assumption of how Ca2+ affects the IP3 receptor. Rather than assuming actual Ca2+ binding sites on the receptor (which are not well characterized), we assume that the actions of Ca2+ arise from its effect on IP3 binding. Thus, although [IP3] is constant during an oscillation, IP3 nevertheless plays an active dynamic role, as oscillations in [Ca2+]i are driven by cycles of IP3 binding and unbinding from the receptor. In the case of CCK-induced oscillations, the receptor also undergoes periodic cycles of phosphorylation and dephosphorylation; the oscillation period is then set by the rate of receptor dephosphorylation, rather than by the rate of receptor recovery from inactivation. Calcium-induced calcium release also plays an important role. As [Ca2+]i increases, the rate of IP3 binding to the receptor is increased (S to O transition), leading to an effective calcium activation of the receptor.
Another major difference from previous models is
our use of the phosphorylation pathway, through which
some agonists will shunt the receptor. It has been recognized for some time that a third mechanism, in addition to Ca2+ activation and inactivation of the IP3R, is
required to explain the long interspike intervals that
occur in some cells (see, for example, Oancea and
Meyer, 1996, for an excellent discussion of this). Indeed, phosphorylation and dephosphorylation of the
IP3R has previously been proposed as a possible mechanism for setting the interspike interval (Cameron et al.,
1995
; Zhu et al., 1996
; in these papers, the phosphorylation was assumed to be Ca2+ dependent, which it does
not appear to be in our cell type). We have shown that
such a hypothesis is quantitatively consistent with the
observed oscillations, we have collected experimental data that show agonist-dependent rates of phosphorylation of the IP3 receptor and have shown that a single
mechanism can be used as a framework to understand
widely differing experimental results in a single cell
type. Ours is not the first model to generate long interspike intervals. Laurent and Claret (1997)
constructed a model of Monod-Wyman-Changeux type and showed
that, with appropriate choices of the parameters, the
model could reproduce long-period oscillations, while
Dupont and Swillens (1996)
postulated the existence of
an intermediate Ca2+ domain around the mouth of the
receptor to achieve a similar result. Our model differs
from these in that we present a single mechanism that
can generate a wide variety of oscillatory patterns, and
then show experimentally that the necessary elements
of the model exist in a particular cell type.
The simplicity of our model can, in some circumstances, be a disadvantage. Our assumption of independent subunits is unlikely to be correct (as pointed out
by Laurent and Claret, 1997), and a more detailed allosteric model of the effects of Ca2+ on IP3 binding
would presumably increase the model's accuracy. However, we believe that the additional complications introduced by such procedures are unlikely to be a worthwhile expenditure of effort at this stage. More detailed
models of the receptor states and phosphorylations can
wait until the properties of the pancreatic acinar IP3 receptors are known in more detail, and until spatial effects are better understood.
Zhu et al. (1996) have presented a qualitative model
that, in many respects, is similar to ours. They conclude
that histamine-stimulated Ca2+ oscillations in HeLa
cells result from cycles of phosphorylation and dephosphorylation of the IP3R by CaMK-II. In their model, just
as in ours, phosphorylation of the IP3R inhibits Ca2+ release by closing the channel. They showed that inhibition of the phosphatase by calyculin A or okadaic acid
(which corresponds to a reduction in k5 in our model)
increases the oscillation period, and this result is reproduced by our model (computations not shown). This
emphasizes the importance of receptor dephosphorylation for setting the period of CCK-induced oscillations.
In addition, our model is easily adapted to simulate
phosphorylation of CaMK-II by letting k4 = c4/(0.54 + c4). This assumes that phosphorylation by CaMK-II is
modulated by Ca2+ in a cooperative fashion, and thus
includes the possibility of feedback from the Ca2+ signal to receptor phosphorylation. In this case, the
model again exhibits long-period baseline spiking (computations not shown).
One principal feature of our model that is supported
only indirectly is the assumption that ACh increases
membrane transport of Ca2+, while CCK increases it to
a lesser extent. In other words, when the cell is at steady
state (i.e., in the absence of agonist stimulation) there
is a continual, but small, flux of Ca2+ through the cytoplasm, as Ca2+ leaks into the cell and is quickly removed. Upon ACh stimulation, overall membrane
transport of Ca2+ is greatly increased, in both directions, and this, in combination with activation of the
IP3R, results in raised-baseline, sinusoidal, oscillations
that are dependent on external Ca2+. CCK, on the
other hand, increases membrane transport of Ca2+
only slightly, and thus CCK-induced oscillations depend on external Ca2+ much less. It has been known
for some years that the rate of Ca2+ turnover is low at
steady state, and that agonists such as CCh and CCK increase the rate of Ca2+ pumping out of the cytoplasm,
as well as increasing the rate of Ca2+ influx (Muallem
and Beeker, 1989; Zhang et al., 1992
). However, what is
not clear is whether ACh increases the rate of Ca2+
turnover more than does CCK. Our model makes the
assumption that it does. Although we used our model
to make predictions about the effects of removing external Ca2+ and subsequently increasing [ACh], and
these predictions were confirmed experimentally, and
although the model provides a consistent framework
that can possibly explain the different effects of low extracellular Ca2+ on ACh- or CCK-induced oscillations,
this is still only indirect evidence for this assumption.
One important test of the model will be to compare
transmembrane and trans-ER Ca2+ fluxes directly, in
the presence of CCK or ACh, or neither.
Lawrie et al. (1993) have shown that application of
ACh in the absence of external Ca2+ will initially cause
oscillations, but these die away within a few minutes.
This particular result is not reproduced by our model
because of the simplified way in which it treats Ca2+
pumping. When Ca2+ is removed from the cytoplasm
by pumps, it can be removed either to the outside or to
the ER. It is easy to see that the rate of removal to the
ER has no effect on the long term steady state [Ca2+]i
of the cell, which can only be affected by Ca2+ transport
across the plasma membrane. When ACh is applied to a cell in low [Ca2+]i, the kinetics of the IP3R can still
lead to cycles of Ca2+ release and uptake from the ER,
but each time Ca2+ is released into the cell cytoplasm, a
fraction of it is lost to the outside. Eventually, the cell
runs down and oscillations stop, as observed. Thus,
over a longer time scale, depletion of the ER plays a
role in terminating Ca2+ oscillations. The only way to
model this is to treat Ca2+ transport across the ER
membrane separately from Ca2+ transport across the
plasma membrane, a feature that is omitted from our
model for the sake of simplicity. However, we have constructed an extended version of the model, in which we
take into account depletion of the ER (LeBeau, Yule,
and Sneyd, unpublished data). The extended model
behaves in a similar manner to the model presented here, but can account for a wider array of experimental
results, including long-time oscillatory behavior and
the application of ACh in the absence of external Ca2+.
Details of this model and comparison with experimental results will be presented in a later paper.
Another important feature of the model is the assumption that an increase in [Ca2+]i causes an increase
in the binding affinity of IP3 to its receptor. The experimental data on the properties of IP3 binding as a function of Ca2+ are not completely consistent. Yoneshima
et al. (1997) claim that Ca2+ increases the binding affinity of IP3 to type-III receptors, but decreases the
binding affinity to type-I receptors. However, Cardy et
al. (1997)
, although agreeing that the major effect of Ca2+ on IP3 binding is stimulatory for type-III and inhibitory for type-I receptors, claim that these effects are
mediated by changes in the maximal binding (although, for type-III receptors, more complex effects occur at higher Ca2+ concentrations). Nevertheless, they
conclude that Ca2+ regulates the interconversion of the
IP3R between two different states, one state with a high
IP3 affinity, and the other with a low affinity. This interpretation is entirely consistent with our results, as our
model can in fact be rigorously derived by considering
just such a receptor mechanism (Sneyd, LeBeau, and Yule, manuscript submitted for publication).
As we show in the Appendix , a decrease in IP3 binding affinity with increasing Ca2+ implies that the peak
of the steady state open probability curve shifts to the
right as [IP3] increases. Conversely, an increase in the
IP3 binding affinity with increasing Ca2+ implies that
the steady state open probability curve will shift to the
left with increasing [IP3]. Thus, since type-I receptors have a decreasing IP3 binding affinity with increasing
Ca2+, the model predicts that the steady state open
probability curve will shift to the right with increasing
[IP3], exactly as seen by Kaftan et al. (1997). On the
other hand, since type-III receptors have an increasing
IP3 binding affinity with increasing Ca2+, we predict
that the steady state open probability curve will shift to
the left with increasing [IP3]. These measurements
have yet to be performed. No models to date (at least
that we know of) have investigated the effects of
changes in the maximal binding. In this context, it is
important to note that the open probability curve at infinite [IP3] does not give the maximal binding curve. In
our model (and all other receptor binding models that
we have investigated), the maximal binding fraction is
always 1 and, as in the presence of large amounts of IP3,
all receptors will bind IP3.
As well as the temporal responses to ACh and CCK
being quite different, the spatial characteristics of the
response are also agonist dependent (Lawrie et al.,
1993; Thorn et al.; 1993a; Xu et al., 1996b
). ACh-
induced oscillations are initiated at the secretory pole
of the cell, and spread from there across the cell, with
the response at the basal pole being consistently of
smaller amplitude and slightly delayed. During the
course of this intracellular wave, significant Ca2+ gradients are maintained in the cell cytoplasm. CCK, on the
other hand, causes an increase in [Ca2+]i that is simultaneous across the entire cell, with no change in amplitude from secretory to basal poles. Furthermore, these intracellular Ca2+ waves can be transmitted intercellularly when individual cells in acini are coupled to their
neighbors via gap junctions (Yule et al., 1996
). A prerequisite for the study of such intra- and intercellular
waves is a detailed understanding of the kinetics underlying the oscillatory response in each individual cell.
Thus, although the current study does not address
these questions, our model provides a useful framework that can be used to study the problem of wave
propagation in this cell type.
![]() |
FOOTNOTES |
---|
Address correspondence to Dr. James Sneyd, Department of Mathematics, University of Michigan, 525 E. University Ave., East Hall, Ann Arbor, MI 48109-1109. Fax: 734-763-0937; E-mail: jsneyd{at}math.lsa.umich.edu
Original version received 1 December 1998 and accepted version received 11 March 1999.
Drs. Yule and Groblewski's present address is Department of Pharmacology and Physiology, University of Rochester, School of Medicine, Rochester, NY. ![]() |
APPENDIX |
---|
The Steady State Open Probability of the IP3R
In the majority of current models of the IP3R, the steady state receptor open probability, O, can be expressed in the general form
![]() |
(11) |
for some functions 1 and
2. The exact form of these
two functions differs from model to model. However,
we note two things. First, p =
2(c) gives the half-maximal IP3 binding to the receptor, and thus
2(c) is the
EC50 of the IP3 receptor, with respect to IP3 binding. It
thus follows from experimental data (Yoneshima et al.,
1997
) that, for type-I receptors,
2 is an increasing function of Ca2+, while for type-III receptors,
2 is a decreasing function of Ca2+. Secondly,
1(c) is the open
probability as a function of c, when p is very large; i.e.,
in the limit of high [IP3].
Bell-shaped Steady State Curve
If O is a bell-shaped function of c, it follows that it must
have a turning point, at which place = 0. Hence, at
the turning point,
![]() |
(12) |
If we let c0 denote the position of the turning point, then Eq. 12 can be solved to obtain c0 = c0(p). We want to know whether c0 is an increasing or decreasing function of p. In other words, does the peak of the bell-shaped curve move to the right or to the left as p increases.
First, we note that an implicit differentiation gives
![]() |
(13) |
It thus follows that the only way c'0(p) can change sign
is if '1(c0) changes sign. [We do not let the denominator change sign, because if it did, then c'0(p) would become infinite, which is clearly unphysiological. For the
same reason, we also exclude singular cases, where
both the numerator and denominator vanish together.]
Finally, we note that, at the turning point, p +
2 =
1
'2/
'1, and so, from Eq. 11, it follows that the open
probability at the turning point, Otp, is given by
![]() |
(14) |
We know that, for type-I receptors, 2 is an increasing
function of c. Hence, for this receptor type,
'2 > 0. From Eq. 14, it now follows that Otp is positive only
when
'1 > 0 at the turning point. However, recall that
1 is just the open probability curve at high p. Hence,
for lower values of p, the peak of the open probability
curve must occur at values of c for which
'1 > 0. Since
'1 cannot change sign, neither can c'0(p). It remains
only to determine which sign it is. However, it is easy to
see that, when p is large, c'0(p) must be positive. Hence,
c'0(p) is always positive for all p, from which it follows
that the peak of the bell-shaped curve must move to the
right as p increases. [It is interesting to note that the
same argument works when
2 is a decreasing function
of c, and thus
1 < 0. In this case, c'0(p) is always negative for all p, from which it follows that the peak of the
bell-shaped curve must move to the left as p increases.)
Monotonic Steady State Curve
Type-III receptors do not have bell-shaped steady state open probability curves, and thus the above argument does not apply to them. However, a similar argument can be used to show that the steady state open probability curve (i.e., the point of maximum slope) shifts to the left as p increases.
Suppose the steady state open probability curve is monotonically increasing, and define
![]() |
(15) |
![]() |
(16) |
For a fixed value of p, the steady state open probability curve, O, reaches half its maximum value at the point c0, where c0 is found by solving the equation
![]() |
(17) |
Note that this defines c0 as a function of p; i.e., c0(p). We now want to find out whether c0 is an increasing function of p (in which case the curves shift to the right as p increases), or a decreasing function of p (in which case the curves shift to the left as p increases).
Differentiate both sides with respect to p to get
![]() |
(18) |
where all functions of c are evaluated at c0.
For a type-III receptor, '2(c) < 0 (as argued above),
and we also suppose that the steady state open probability curve is monotonic for all IP3 concentrations and thus
'1(c) > 0. If, for any p, the steady state curve becomes
bell-shaped, the previous argument applies. Since
![]() |
(19) |
it now follows that <
2(c), and
>
1(c), and
thus c '0( p) < 0.
In conclusion, we have predicted that, as p increases, the steady state open probability curve of a type-III IP3R will move to the left. To our knowledge, this has not yet been experimentally measured.
We thank John Williams for helpful discussions. We also thank Dr. Jean-François Dufour, The American Society for Biochemistry and Molecular Biology Inc., and Cell Calcium for permission to reprint figures.
This work was supported by grants from the New Zealand Lottery Grants Board (AP047957), the Marsden Fund of the Royal Society of New Zealand, NIGMS grant R01 GM56126 (J. Sneyd), National Science Foundation grant DMS 9706565 (JS) and National Institute of Diabetes and Digestive and Kidney Diseases grant R01 DK54568 (D.I. Yule).
![]() |
Abbreviations used in this paper |
---|
Ach, acetylcholine; CaMK-II, Ca2+-calmodulin kinase II; CCK, cholecystokinin; CPA, cyclopiazonic acid; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor.
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