From the * Department of Physiology, and Department of Medicine, University of Connecticut, Farmington, Connecticut 06030-3505
The inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751-754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443-446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642-29649; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591-598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 µM InsP3, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751-754). We report here that, when we used lower InsP3 concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP3 concentrations, channel activity persisted at Ca concentrations as high as 30 µM. To explore this unexpected response of the channel, we measured InsP3 binding over a broad range of InsP3 concentrations. We found the well-characterized high affinity InsP3 binding sites (with Kd < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61-67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192-195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468-470) and a low affinity InsP3 binding site (Kd = 10 µM). Using these InsP3 binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP3 levels and the maintained channel activity at high Ca and InsP3 levels. The observed Ca dependence of the InsP3-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP3, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.
Key words: cerebellum; ligand binding; intracellular calcium channel; channel regulationThe inositol 1,4,5-trisphosphate (InsP3)1 receptor is an
intracellular calcium (Ca) release channel found in virtually all cell types (Berridge, 1993; Bezprozvanny and
Ehrlich, 1995
; Clapham, 1995
; Divecha and Irvine,
1995
). Activation of the InsP3-gated channel causes an
increase in cytoplasmic Ca by releasing Ca from the endoplasmic reticulum. InsP3-mediated Ca release is important for many cellular processes, including the expression of transcription factors (Negulescu et al., 1994
),
the formation of the fertilization envelope during egg
activation (Nuccitelli et al., 1993
), nuclear membrane
reformation in mitosis (Sullivan et al., 1995
), stimulus-contraction coupling in smooth muscle (Walker et al.,
1987
), and the development of long term depression
(Kasono and Hirano, 1995
).
The InsP3-gated channel exists as a complex comprised of four subunits of 260 kD each. To date, three
isoforms of the subunits have been cloned (Furuichi et
al., 1989; Mignery et al., 1989
; Sudhof et al., 1991
; Blondel
et al., 1993
; Maranto, 1994
; Morgan et al., 1996
). The
isoform type and extent of expression is cell-type specific. The cerebellar Purkinje cell expresses almost exclusively type 1 receptor at levels at least 10× greater
than other cell types. Hepatocytes express both type 1 and type 2 receptors, pancreatic acinar cells express
type 2 and type 3 receptors, and several epithelia express
all three receptor types (Bush et al., 1994
; Nathanson et
al., 1994
; Wojcikiewicz, 1995
). Further diversity may exist in tissues where different isoforms associate to form heterotetramers (Joseph et al., 1995
; Monkawa et al.,
1995
).
Each subunit of the tetrameric channel complex contains an InsP3 binding site near the NH2 terminus
(Mignery and Sudhof, 1990). InsP3 binds to InsP3-gated
channels from cerebellum with high affinity (Kd ranging from 5 to 50 nM). An additional low affinity site for
InsP3 was described using the InsP3 analog InsP3S3, but the location of the site was unclear because crude microsomes were used (Challiss et al., 1991
). Ca inhibits
InsP3 binding to the InsP3-gated channel (Worley et al.,
1987
; Danoff et al., 1988
). The Ca-dependent inhibition of InsP3 binding can be reversibly removed by purifying the channel by heparin affinity chromatography, suggesting that Ca sensitivity is conferred by an accessory protein (Danoff et al., 1988
).
InsP3 is the only known physiological activator of the
InsP3-gated channel. Activation of the channel with
InsP3 is reversible: channels stop opening if InsP3 is
washed out and readdition of InsP3 reactivates the
channels (Ehrlich and Watras, 1988). In the presence
of InsP3, Ca acts as an allosteric regulator of the InsP3-gated Ca channel (Iino, 1990
; Bezprozvanny et al., 1991
; Finch et al., 1991
; Hannaert-Merah et al., 1994
; Marshall and Taylor, 1994
). Previous studies using permeabilized smooth muscle (Iino, 1990
), Ca release from
cerebellar microsomes (Finch et al., 1991
), and single
channel recordings (Bezprozvanny et al., 1991
) showed
that the Ca dependence of channel activity is described by a bell-shaped curve. With single channel recordings,
maximal channel activity was observed in the presence
of 0.25 µM free Ca and there was a steep decline in
channel activity on either side of the maximum (Bezprozvanny et al., 1991
). Complete inhibition of channel activity occurred when cytoplasmic Ca reached 5 µM. The Ca-dependent activation provides an amplification of the initial signal and the Ca-dependent inhibition of the InsP3-gated channel allows for fast negative
feedback of the cytoplasmic Ca concentration.
A number of models have been proposed to describe
the regulation of the InsP3-gated channel. Most models
assume three regulatory sites on the channel: one site
for InsP3, one site for activating Ca, and one site for inhibitory Ca. Under steady state conditions, these models make different predictions of the shape of the Ca-dependence curve as InsP3 concentrations are varied.
As the InsP3 concentration is increased, some models
predict that the peak of the Ca-dependence curve will
(a) shift to lower Ca concentrations (Othmer and Tang, 1993), (b) shift to higher Ca concentrations (De Young
and Keizer, 1992
), or (c) remain unchanged (Atri et al.,
1993
; Bezprozvanny and Ehrlich, 1994
).
In this paper, we measured the Ca dependence of InsP3-gated channel activity as a function of InsP3 concentration. We also compared InsP3 binding and channel activity using the same experimental conditions. Using our measured values, we constructed a model that accounts for the interaction of Ca and InsP3 in regulating the InsP3 receptor. The model is distinct from other models currently applied to the InsP3-gated Ca channel and is consistent with the observed leftward shift of the curve at low InsP3 concentrations. A novel feature of the model is the inclusion of a low affinity InsP3 binding site, which broadens the range of regulation of the channel by InsP3 and explains the observed maintained activity of the channel at high concentrations of Ca and InsP3.
Single Channel Recordings
Canine cerebellar endoplasmic reticulum vesicles were prepared
as previously described (Ehrlich and Watras, 1988) and fused with planar lipid bilayers composed of phosphatidylethanolamine and phosphatidylserine (3:1, wt:wt; Avanti Polar Lipids, Alabaster, AL) dissolved in decane (20 mg/ml). Cytoplasmic bilayer solutions contained 500 µM ATP, 500 µM EGTA, 110 mM Tris, and
250 mM HEPES, pH 7.35, and luminal solutions contained 53 mM Ba(OH)2, 250 mM HEPES, pH 7.35. Calibrated CaCl2 was
added to the cytoplasmic solution to obtain the desired free Ca
concentration (Fabiato, 1988
). Because estimation of free Ca was
critical, calculations were routinely checked spectrofluorometrically with BTC (Molecular Probes, Inc., Eugene, OR). The number of channels in each experiment was estimated from the maximum number of channels observed simultaneously in the bilayer
(Horn, 1991
). The InsP3 dependence of the open probability,
measured at a fixed Ca concentration, was used to correct for
variations in the maximum open probability among individual channels. Transmembrane voltage was maintained at 0 mV and
the single channel current amplified (Warner Instruments, Hamden, CT) and stored on VHS tape (Instrutech Corp., Great Neck,
NY). Data were filtered at 1 kHz and digitized at 5 kHz for computer analysis using pClamp 6.0 (Axon Instruments, Foster City, CA).
InsP3 Binding Measurements
[3H]InsP3 binding was measured as previously described (Benevolensky et al., 1994) except that binding was measured using solutions similar to the cytoplasmic solution in single channel experiments (500 µM ATP, 500 µM EGTA, 110 mM Tris, 250 mM
HEPES, pH 7.35, with the specified free Ca concentration). Radioligand concentrations were varied from 0.4 nM to 30 µM. To
achieve this range, stock radioligand (480 nM; New England Nuclear, Boston, MA) was diluted with unlabeled InsP3 (Calbiochem,
La Jolla, CA). To assure sufficient reliability in the measurement,
the protein concentration was increased as the specific activity
decreased. Nonspecific binding was measured in the presence of
2.5 mM unlabeled InsP3 or 1-10 mg/ml heparin. These conditions generated the same value for nonspecific binding. To be included in the analysis, specific binding had to exceed 50% of the
nonspecific binding. As Scatchard analysis of InsP3 binding showed
the presence of three binding sites, InsP3 binding was modeled as
shown:
![]() |
(1) |
where KH, KM, and KL are the apparent dissociation constants for the 1-nM, 50-nM, and 10-µM sites and bH, bM, and bL refer to the maximum binding at the respective sites.
In most published reports, InsP3 binding experiments were done using conditions that maximize binding, pH 8.0, at 4°C. In contrast, measurements of InsP3-gated channel function have generally been done using conditions closer to physiological conditions, pH 7.3, at 22-37°C. In the experiments described here, measurements were done using similar conditions when possible to compare in vitro channel function with biochemical properties. In addition, InsP3 saturation binding curves were generated at 0 and 22°C to determine the temperature coefficient of binding (Q10). The Q10 was determined to be 2.3 for the 50-nM site and was estimated to be 1.0 for the 10-µM site. Although the Q10 value for the 10-µM site is consistent with our data, it must be called an estimate due to low signal-to-noise ratio in binding measurements at 22°C in combination with the low specific activity obtained when very high InsP3 concentrations are used.
Modeling of Channel Function Using Single Channel Data and Binding
The "2-IP3/2-Ca" model used for the analysis of the open probability data assumes that the InsP3-gated Ca channel complex contains four monomers, and that each monomer of the tetrameric channel complex has two InsP3 binding sites (with apparent dissociation constants K50nM and K10µM), one Ca binding site for activation of the channel (CaAC), and one Ca binding site for inhibition of the channel (CaIN). The affinity of the 50-nM site depends upon the occupancy of the CaIN site, as determined from binding experiments in the presence and absence of Ca (see Fig. 3 C and Benevolensky et al., 1994). To fit the model to the data, it was not
necessary to include cooperativity of binding among the sites or
sequential binding steps.
The presence of four ligands associated with each monomer means there are 16 possible states (24) of each monomer of the tetrameric Ca channel complex. To fit the open probability data over a wide range of InsP3 and Ca concentrations, we had to assume that more than 1 of the 16 states was able to conduct Ca. The simplest model that fit the open probability data and the InsP3 binding data required that 3 of the 16 possible states be capable of conducting Ca. The three possible conducting states are: S1 = InsP3 bound to the K50nM site, and Ca bound to the CaAC site; S2 = InsP3 bound to the K50nM and K10µM sites, and Ca bound to the CaAC site; and S3 = InsP3 bound to the K50nM and K10µM sites, and Ca bound to the CaAC and CaIN sites.
The relative abundance (RA) of the three possible conducting states is determined as follows:
![]() |
(2) |
where ai is the calculated fractional abundance of the ith state of the channel based on equilibrium binding constants of the transitions among the 16 states, assuming mass action kinetics. Then, the single channel open probability (Po) is calculated as follows:
![]() |
(3) |
where Pi is the probability over time that the channel complex will open assuming that i of the monomers in the channel complex are in one of the conducting states. An iterative curve fitting routine (Sigmaplot; Jandel Scientific, San Rafael, CA) was used to calculate the equilibrium constants of the various transitions that best fit both the single channel data and the InsP3 binding data.
InsP3 Shifts the Bell-shaped Calcium-dependence Curve
The Ca dependence of the InsP3-gated Ca channel was
previously monitored at the single channel level using
2 µM InsP3, a concentration 10× the Kd previously determined for InsP3-gated release and channel activity
(Watras et al., 1991). Measurements of InsP3-dependent Ca release from vesicles showed that inhibition by
Ca varied with InsP3 concentration (Joseph et al., 1989
;
Combettes et al., 1994
; Bootman et al., 1995
; Hannaert-Merah et al., 1995
). Specifically, as the InsP3 concentration was increased, Ca-dependent inhibition of InsP3-induced Ca release occurred at higher Ca concentrations (Joseph et al., 1989
; Combettes et al., 1994
; Bootman
et al., 1995
; Hannaert-Merah et al., 1995
). We now report a similar response at the single channel level (Fig.
1). In the presence of either 0.2 or 2 µM InsP3, cytoplasmic Ca activates single channel currents over a similar concentration range (compare Fig. 1, A and B, top 3 traces). As the InsP3 concentration is elevated, activity is maintained at higher cytoplasmic Ca concentrations
(Fig. 1, A and B, bottom 4 traces). A comparison of the
open probability of the InsP3-gated channel as a function of cytoplasmic Ca concentration shows that the
peak channel activity shifts from 0.1 to 0.25 µM Ca
when the InsP3 concentration is increased from 0.2 to 2 µM (Fig. 2,
and
). However, the activating phase of
each curve is quite similar. The predominant effect of
raising the InsP3 concentration is an increase in channel activity above 0.1 µM Ca. For example, the channel
is essentially closed at 1 µM free Ca in the presence of
0.2 µM InsP3, but there is substantial channel activity in
the presence of 2 µM InsP3.
If the InsP3 concentration is increased further, an unexpected response of the channel is observed. At 180 µM InsP3, channel activity remains robust at all Ca concentrations tested (Fig. 1 C). Note that the activity measured at 30 µM Ca is essentially the same as that observed at 5 µM Ca (Fig. 2, ). The relatively large open
probability of the channel in the presence of 5-30 µM
Ca and high InsP3 concentrations was not predicted in
published models (De Young and Keizer, 1992
; Bezprozvanny, 1994
; Tang et al., 1996
) of the regulation of
the InsP3-gated channel by Ca and InsP3. The persistent
activity of the channel at high levels of InsP3 provides a
means for the cell to maintain intracellular Ca beyond
1 µM during periods of continued stimulation of the phosphoinositide cascade.
Analysis of [3H]InsP3 Binding Reveals a Low Affinity Site
To investigate the persistent elevation of channel activity in the presence of high concentrations of Ca and
InsP3, a series of InsP3 binding experiments were undertaken. When InsP3 concentrations spanning a broad
range are used (0.4 nM to 30 µM), the binding data
shows three distinct slopes indicating at least three
binding sites for InsP3 (Fig. 3 A). Three sites are evident in all four cerebellar preparations tested. The
InsP3-gated Ca channel purified by either immunoprecipitation with a type 1-specific InsP3 receptor antibody
or by heparin-sepharose column chromatography (Fig.
3 B) also had three distinct binding sites, which indicates that these sites are integral parts of the channel
complex. The high affinity (<1 nM) binding site is least
abundant, representing <1% of the total sites in all
preparations tested. This high affinity site has been observed previously in cerebellum and vascular smooth muscle (Hingorani and Agnew, 1992; Benevolensky et
al., 1994
). Two binding sites of approximately equal
abundance account for the remaining 99% of the InsP3
binding in this tissue; the Kd's of the sites when measured at 0°C are 54 nM and 10.2 µM. In this text, these
InsP3 binding sites are called the 1-nM, 50-nM, and 10-µM
sites. The 50-nM site is the InsP3 binding site that has been shown previously to be concentrated in cerebellar
Purkinje cells (Mignery et al., 1989
; Ross et al., 1989
;
Maeda et al., 1990
). We report here the existence of a
low affinity site that saturates above 10 µM InsP3 (Fig. 3,
A and B). The Kd of this site is more than 200× higher
than those previously shown for the purified InsP3 receptor. The 10-µM site identified in this report may be
the site responsible for low affinity InsP3 binding described indirectly in crude cerebellar microsomes (Challiss et al., 1991
).
To investigate the inositol phosphate specificity of
the two predominant InsP3 binding sites, competition
binding was done at two concentrations of InsP3, 10 nM
[3H]InsP3 to examine the 50-nM site and 6 µM [3H]InsP3
to examine the 10-µM site (Fig. 4). For each concentration of InsP3, three competitors were examined:
1,3,4,5-InsP4, 1,4,5-InsP3, and 2,4,5-InsP3. At the 50-nM
site, 1,4,5-InsP3 is at least 30× more effective than
1,3,4,5-InsP4 at displacing 10 nM [3H]1,4,5-InsP3 from
the receptor (Fig. 4 A). The ability of 2,4,5-InsP3 and
1,3,4,5-InsP4 to displace 30 nM [3H]1,4,5-InsP3 from
the 50-nM site was indistinguishable. Thus, 1,4,5-InsP3
has the highest affinity at the 50-nM site and both 2,4,5-InsP3 and 1,3,4,5-InsP4 have 30-fold lower affinity for this site. In contrast, 1,3,4,5-InsP4 appears more effective than 1,4,5-InsP3 in its ability to displace 6 µM
[3H]1,4,5-InsP3 from the 10-µM site and 6 µM 2,4,5-InsP3 was unable to displace [3H]1,4,5-InsP3 from the
10-µM site (Fig. 4 B). Therefore, the 10-µM site can be
distinguished from the 50-nM site by its relative specificity for inositol phosphates.
The possibility that other isoforms of the InsP3 receptor were responsible for the 10-µM site was ruled out by
comparing the amount of the three InsP3 receptor isoforms in both purified preparations using Western
analysis. We obtained strong staining for type 1 and, despite using 10× more protein, virtually no type 2 or 3 InsP3 receptors, confirming published reports that cerebellum contains >90% type 1 InsP3 receptor (Sudhof
et al., 1991; Wojcikiewicz, 1995
; Morgan et al., 1996
).
These values need to be compared with the observation
that the 50-nM and 10-µM sites are present in approximately equal abundance.
Micromolar concentrations of Ca alter InsP3 binding
in a variety of tissues (Pietri et al., 1990; Marshall and
Taylor, 1994
; Watras et al., 1994
). In cerebellum and
vascular smooth muscle, Ca decreases InsP3 binding
(Worley et al., 1987
; Danoff et al., 1988
; Benevolensky
et al., 1994
). Both of these tissues contain predominantly the type 1 InsP3 receptor (Marks et al., 1990
; Furuichi et al., 1993
; Newton et al., 1994
). We find that
InsP3 binding to cerebellar membranes under conditions identical to those used for the single channel
measurements also shows Ca-dependent inhibition (Fig.
3 C). Only binding to the 50-nM site appears to be Ca
sensitive. Elevation of Ca to 10 µM decreases, but does
not completely inhibit, InsP3 binding (Fig. 3 C,
).
Similarly, in vascular smooth muscle, Ca concentrations as high as 150 µM failed to completely inhibit InsP3 binding (Benevolensky et al., 1994
). In contrast,
InsP3 binding to both the 1-nM and 10-µM sites appears insensitive to Ca (Fig. 3 C).
Model of InsP3-gated Channel Function Needs the Novel InsP3 Binding Site
We created a model of InsP3-gated channel function
that accounts for the Ca dependence of InsP3 binding
(Fig. 3) and the Ca dependence of channel activity
(Fig. 2) over a broad range of InsP3 concentrations.
This model is named the 2-InsP3/2-Ca model because it
incorporates two InsP3 binding sites (50 nM and 10 µM) and two calcium regulatory sites (activating and
inhibitory) on each monomer of the tetrameric channel complex. The 1-nM site for InsP3 is not included in
the model due to its low abundance. A model incorporating one InsP3 site and two Ca sites previously proposed (De Young and Keizer, 1992) is able to predict
channel activity when InsP3 levels are <2 µM, but the
one InsP3 binding site model requires parameters that
are inconsistent with InsP3 binding data. In addition,
this model with one InsP3 binding site cannot predict
the persistent activity observed at high concentrations of InsP3 and Ca. In our 2-InsP3/2-Ca model, InsP3 binding to the 10-µM site allows channel activity to be sustained at cytoplasmic Ca concentrations above 5 µM.
Values for the affinity of InsP3 in the absence of Ca were determined from binding experiments (Fig. 3); other parameters were predicted from fits of the model to both the single channel and binding data (Table I). All curves through the experimental points (Figs. 2 and 3) were generated by the 2-InsP3/2-Ca model. Using the parameters generated by the fit of the model to the data, the open probability of the channel can be predicted over a wide range of both InsP3 and Ca. Predictions of channel activity at concentrations of InsP3 and Ca up to 1 mM are shown in Fig. 5.
Table I. "2-InsP3/2-Ca" Model Parameters |
An outcome of the 2-IP3/2-Ca model is that at least
two of the four monomers of the tetrameric Ca channel
complex must be in one of the three possible conducting states for the channel complex to conduct Ca. That
is, if only one of the monomers in the tetrameric complex is in one of the conducting states (P1 in Eq. 3), the
predicted value for the probability that the channel will
open was 1011, suggesting that the singly occupied
channel rarely opens. In contrast, if two or three monomers are occupied, the predicted values for the probability that the channel will open were 0.06 and 0.04, respectively. The requirement for at least two InsP3 molecules to bind to the receptor is supported by experimental findings. An extension of our earlier experiments describing the InsP3 concentration dependence
of the open probability of the channel (Watras et al.,
1991
) to lower concentration of InsP3 (10 nM, data not
shown) generates a curve with a Hill coefficient of 1.8. This result and additional reports (Somlyo et al., 1992
; Marchant et al., 1997
) support the suggestion that multiple molecules of InsP3 bind to the channel before it
opens.
As a further test of the model, single channel behavior was measured at 20 nM InsP3 (Fig. 2, ). At this very
low InsP3 concentration, channel activity was difficult
to measure because openings were infrequent, but the
shape of the Ca dependence and the peak in channel
activity were similar to the predicted values. Although other models could be generated, the ability of the
2-InsP3/2-Ca model to fit both the single channel and
binding data and to predict InsP3-gated channel function under a variety of conditions lends support for this
model of channel function.
In this paper, we tested the effect of changing the InsP3
concentration on the regulation of the InsP3 receptor.
We found that the peak of channel activity shifted to
higher Ca concentrations as the InsP3 concentration
was increased from 20 nM to 2 µM and that elevating
the InsP3 concentration above 2 µM leads to persistent activation of the InsP3-gated channel. To explain this
unexpected response, we measured InsP3 binding at
InsP3 concentrations from 0.4 nM to 30 µM. We found
the well-characterized high affinity InsP3 binding sites
(with Kd's < 1 and 50 nM) (Mignery et al., 1989; Ross et
al., 1989
; Maeda et al., 1990
) and a low affinity InsP3
binding site (Kd = 10 µM). We then developed a new
model that accounts for both the channel activity and
the InsP3 binding properties over the entire range of
InsP3 and Ca concentrations tested.
We measured a large increase in steady state channel
activity by elevating the InsP3 concentration (Fig. 1). It
is interesting to note that these changes occurred in
the absence of other cellular processes that have been
implicated in the regulation of InsP3-gated channel activity. For example, it has been proposed that the activating phase of the calcium dependence curve relies
upon phosphorylation of the channel by protein kinase
C and that the inhibitory phase of the calcium-dependence curve reflects the dephosphorylation of the channel by calcineurin (Cameron et al., 1995). In this series
of experiments, the increase in channel activity is unlikely to be attributed to phosphorylation because Mg-ATP was not present (Na-ATP was used in the experimental protocol) and no kinase was added to the system.
Similarly, it is unlikely that the decrease in channel activity measured in these experiments is the consequence of calcineurin activity because, in preliminary
experiments, we were unable to detect calcineurin in
our microsomal preparation. The ability to reverse the
effects of elevated Ca in the absence of added kinase or
Mg-ATP in our experiments also argues against an absolute requirement for phosphorylation/dephosphorylation in the Ca-dependent regulation of InsP3-gated
channel activity.
The mechanism underlying the Ca-dependent inhibition of InsP3 binding to the 50-nM InsP3 binding site
is unclear, but may involve the presence of an accessory
protein associated with the InsP3 receptor (Danoff et
al., 1988; Ferris and Snyder, 1992
; Benevolensky et al.,
1994
). Our experiments suggest that the 10-µM site
cannot reside on the associated protein that confers Ca
sensitivity to the InsP3 receptor. The InsP3 receptor purified with heparin affinity chromatography is calculated to be 94% pure and it lacks Ca sensitivity. The
heparin affinity purified channel, however, does retain
the 10-µM site in an approximately one-to-one stoichiometry with the 50-nM InsP3 binding site. Other proteins thought to associate with the InsP3 receptor do
not appear to bind InsP3 (e.g., FKBP12, calcineurin),
strongly implying that the 10-µM site resides on the
InsP3 receptor.
Two properties of the 10-µM site are crucial for
InsP3-gated channel function. First, binding of InsP3 to
the 10-µM site is not Ca dependent (Fig. 3). This allows
InsP3 to remain bound to the receptor even in the presence of high Ca. Indeed, even in the presence of 150 µM Ca it was not possible to remove all of the InsP3
(Benevolensky et al., 1994). With this site, the channel can remain open even when cytoplasmic Ca is at micromolar concentrations. Second, the existence of a binding site with affinity orders of magnitude different from
previously identified binding sites provides a wider dynamic range over which the channel can function.
Three models have been proposed for the regulation
of the InsP3-gated Ca channel (De Young and Keizer,
1992; Atri et al., 1993
; Othmer and Tang, 1993
; Bezprozvanny and Ehrlich, 1994
). Only one of these models (De Young and Keizer, 1992
), however, predicts a
rightward shift in the Ca dependence as the InsP3 concentration is increased from 0 to 2 µM. This model included only one binding site for InsP3 and it gave reasonable fits of our single channel data over the range of
InsP3 concentrations from 0 to 2 µM. However, this
model was unable to fit our InsP3 binding data over the
same range of InsP3 concentrations with the parameters used to fit the single channel data. Moreover, the
one InsP3 binding site model cannot explain the persistence of channel activity at very high concentrations of
InsP3 and high levels of Ca.
When InsP3-induced Ca release was measured in
Purkinje cells of rat cerebellar slices, the cytoplasmic
free Ca was elevated to 26 µM (Khodakhah and Ogden,
1995), well beyond the Ca concentration one would
have initially expected for InsP3-mediated Ca release. Indeed, it was thought that this could never happen because the InsP3-gated channel would be closed by 5 µM
cytoplasmic Ca. The data in the present paper show
that intracellular Ca in the tens of micromolar range
could be achieved by an InsP3-dependent pathway. Moreover, the ability of the channel to remain open at
high intracellular Ca occurs when InsP3 concentrations
are elevated, which is consistent with the need for InsP3
concentrations of 9 µM and higher to induce Ca release in intact Purkinje cells (Khodakhah and Ogden,
1993
, 1995
). Thus, the values for intracellular Ca predicted by the model presented here (see Fig. 5) are
within the range found in intact Purkinje cells.
The interaction between InsP3 and Ca in the regulation of the InsP3-gated channel (Fig. 2) also may explain the pattern of cytoplasmic Ca oscillations evoked
by InsP3 in pancreatic acinar cells. These cells generate
Ca oscillations in the continued presence of low concentrations of InsP3. The oscillations are a consequence, at least in part, of the Ca-dependent inhibition of Ca release through the InsP3-gated channel. When InsP3 levels are 50 µM, sustained elevations in cytoplasmic Ca
are observed (Wakui et al., 1989
; Petersen et al., 1991
).
The ability to sustain an elevation in intracellular Ca in
the presence of high concentrations of InsP3 is consistent with the response of the InsP3-gated channel seen
in the presence of high concentrations of InsP3 (Fig. 2 C).
High concentrations of InsP3 normally exist in a number of cell types under basal (0.1-3 µM InsP3) and agonist-induced (1-20 µM InsP3) conditions (Putney, 1990).
These values for intracellular InsP3 concentrations may
actually be underestimates when confined portions of
the cell, such as dendrites, are considered. That many
cell types have resting and stimulated concentrations of InsP3 thought to be super saturating now has a purpose, to provide a prolonged elevation of intracellular
Ca and to provide a larger dynamic range for InsP3-mediated Ca signaling.
In summary, we show that the interactions between
Ca and InsP3 with the InsP3-gated channel are complex
(Fig. 5). The ability of Ca to regulate the activity of the
InsP3 receptor within the expected range of intracellular Ca concentrations by interacting directly with the
channel complex has been useful for understanding Ca
signaling, waves, and oscillations (Allbritton and Meyer,
1993; Berridge, 1993
; Bezprozvanny, 1994
; Clapham,
1995
). The presence of the newly identified InsP3 binding site on the purified InsP3 receptor provides physiological relevance for the seemingly high levels of basal
(0.1-3 µM) and agonist-induced InsP3 concentrations (1-20 µM; Putney, 1990
). Our results and model show
that more complex interactions among the regulatory
ligands are needed to explain InsP3-gated channel function. The expanded relationship between InsP3 and Ca
demonstrated in the present paper is of great functional
importance as a cell is able to overcome Ca-induced channel inhibition during sustained stimulation by producing more InsP3.
Address correspondence to Dr. E.J. Kaftan, Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195. E-mail: kaftan{at}u.washington.edu
Received for publication 16 July 1997 and accepted in revised form 9 September 1997.
B.E. Ehrlich's present address is Department of Pharmacology, Yale University, New Haven, CT 06510. E.J. Kaftan's present address is Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.The authors thank William Dyckman for his assistance in obtaining canine cerebella. We thank Drs. Joel Brown, Knox Chandler, Larry Cohen, and Frank Striggow for critical comments on the manuscript. Dr. Greg Mignery graciously provided antibody specific for the type 2 InsP3 receptor. We thank Dr. Ion Moraru for invaluable discussions regarding the modeling.
This work was supported by National Institutes of Health grants HL-33026 and GM-51480.
InsP3, inositol 1,4,5-trisphosphate.
1. | Allbritton, N.L., and T. Meyer. 1993. Localized calcium spikes and propagating calcium waves. Cell Calcium. 14: 691-697 [Medline]. |
2. | Atri, A., J. Amundson, D. Clapham, and J. Sneyd. 1993. A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte. Biophys. J. 65: 1727-1739 [Abstract]. |
3. | Benevolensky, D., I. Moraru, and J. Watras. 1994. Micromolar calcium reduces the affinity of the inositol 1,4,5-trisphosphate receptor in smooth muscle. Biochem. J. 299: 631-636 [Medline]. |
4. | Berridge, M.J.. 1993. Inositol trisphosphate and calcium signalling. Nature (Lond.). 361: 315-325 [Medline]. |
5. | Bezprozvanny, I.. 1994. Theoretical analysis of calcium wave propagation based on inositol (1,4,5)-trisphosphate (InsP3) receptor functional properties. Cell Calcium. 16: 151-166 [Medline]. |
6. | Bezprozvanny, I., and B. Ehrlich. 1994. Inositol (1,4,5)-trisphosphate gated Ca channels from canine cerebellum: divalent cation conduction properties and regulation by intraluminal Ca. J. Gen. Physiol. 104: 821-856 [Abstract]. |
7. | Bezprozvanny, I., and B.E. Ehrlich. 1995. The inositol 1,4,5-trisphosphate (InsP3) receptor. J. Membr. Biol. 145: 205-216 [Medline]. |
8. | Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature (Lond.). 351: 751-754 [Medline]. |
9. |
Blondel, O.,
J. Takeda,
H. Janssen,
S. Seino, and
G.I. Bell.
1993.
Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, gastrointestinal tract, and other tissues.
J. Biol. Chem.
268:
11356-11363
|
10. | Bootman, M.D., L. Missiaen, J.B. Parys, H. DeSmedt, and R. Casteels. 1995. Control of inositol 1,4,5-trisphosphate-induced Ca2+ release by cytosolic Ca2+. Biochem. J. 306: 445-451 [Medline]. |
11. |
Bush, K.T.,
R.O. Stuart,
S.H. Li,
L.A. Moura,
A.H. Sharp,
C.A. Ross, and
S.K. Nigam.
1994.
Epithelial inositol 1,4,5-trisphosphate receptors: multiplicity of localization, solubility, and isoforms.
J.
Biol. Chem.
269:
23694-23699
|
12. | Cameron, A.M., J.P. Steiner, D.M. Sabatini, A.I. Kaplin, L.D. Walensky, and S.H. Snyder. 1995. Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. Proc. Natl. Acad. Sci. USA. 92: 1784-1788 [Abstract]. |
13. | Challiss, R.A.J., S.M. Smith, B.V.L. Potter, and S.R. Nahorski. 1991. D- < S-35(U) > inositol 1,4,5-trisphosphorothioate, a novel radioligand for the inositol 1,4,5-trisphosphate receptor. Complex binding to rat cerebellar membranes. FEBS Lett. 281: 101-104 [Medline]. |
14. | Clapham, D.E.. 1995. Calcium signaling. Cell. 80: 259-268 [Medline]. |
15. |
Combettes, L.,
Z. Hannaert-Merah,
J.F. Coquil,
C. Rousseau,
M. Claret,
S. Swillens, and
P. Champeil.
1994.
Rapid filtration studies of the effect of cytosolic Ca2+ on inositol 1,4,5-trisphosphate-induced 45Ca2+ release from cerebellar microsomes.
J. Biol.
Chem.
269:
17561-17571
|
16. | Danoff, S.K., S. Supattapone, and S.H. Snyder. 1988. Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium. Biochem. J. 254: 701-705 [Medline]. |
17. | De Young, G.W., and J. Keizer. 1992. A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agonist-stimulated oscillations in Ca concentration. Proc. Natl. Acad. Sci. USA. 89: 9895-9899 [Abstract]. |
18. | Divecha, N., and R.F. Irvine. 1995. Phospholipid signaling. Cell. 80: 269-278 [Medline]. |
19. | Ehrlich, B.E., and J. Watras. 1988. Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature (Lond.). 336: 583-586 [Medline]. |
20. | Fabiato, A.. 1988. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 157: 378-417 [Medline]. |
21. | Ferris, C.D., and S.H. Snyder. 1992. Inositol 1,4,5-trisphosphate-activated calcium channels. Annu. Rev. Physiol. 54: 469-488 [Medline]. |
22. | Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release. Science (Wash.). 252: 443-446 . [Medline] |
23. | Furuichi, T., D. Simon-Chazottes, I. Fujino, N. Yamada, M. Hasegawa, A. Miyawaki, S. Yoshikawa, J. Guenet, and K. Mikoshiba. 1993. Widespread expression of inositol 1,4,5-trisphosphate receptor type 1 gene (Insp3r1) in the mouse central nervous system. Receptors Channels. 1: 11-24 [Medline]. |
24. | Furuichi, T., S. Yoshikawa, A. Miyawaki, K. Wada, N. Maeda, and K. Mikoshiba. 1989. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature (Lond.). 342: 32-38 [Medline]. |
25. | Hannaert-Merah, Z., L. Combettes, J.-F. Coquil, S. Swillens, J.-P. Mauger, M. Claret, and P. Champeil. 1995. Characterization of the co-agonist effects of strontium and calcium on myo-inositol trisphosphate-dependent ion fluxes in cerebellar microsomes. Cell Calcium. 18: 390-399 [Medline]. |
26. |
Hannaert-Merah, Z.,
J.F. Coquil,
L. Combettes,
M. Claret,
J.P. Mauger, and
P. Champeil.
1994.
Rapid kinetics of myo-inositol
trisphosphate binding and dissociation in cerebellar membranes.
J. Biol. Chem.
269:
29642-29649
|
27. | Hingorani, S.R., and W.S. Agnew. 1992. Assay and purification of neuronal receptors for inositol 1,4,5-trisphosphate. Methods Enzymol. 207: 573-591 [Medline]. |
28. | Horn, R.. 1991. Estimating the number of channels in patch recordings. Biophys. J. 60: 433-439 . |
29. | Iino, M.. 1990. Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. J. Gen. Physiol. 95: 1103-1122 [Abstract]. |
30. |
Joseph, S.K.,
C. Lin,
S. Pierson,
A.P. Thomas, and
A.R. Maranto.
1995.
Heteroligomers of type-I and type-III inositol trisphosphate
receptors in WB rat liver epithelial cells.
J. Biol. Chem.
270:
23310-23316
|
31. | Joseph, S.K., H.L. Rice, and J.R. Williamson. 1989. The effect of external calcium and pH on inositol trisphosphate-mediated calcium release from cerebellum microsomal fractions. Biochem. J. 258: 261-265 [Medline]. |
32. | Kasono, K., and T. Hirano. 1995. Involvement of inositol trisphosphate in cerebellar long-term depression. Neuroreport. 6: 569-572 [Medline]. |
33. | Khodakhah, K., and D. Ogden. 1995. Fast activation of inositol trisphosphate-evoked Ca2+ release in rat cerebellar Purkinje neurones. J. Physiol. (Camb.) 487: 343-358 [Abstract]. |
34. | Khodakhah, K., and D. Ogden. 1993. Functional heterogeneity of calcium release by inositol trisphosphate in single Purkinje neurones, cultured cerebellar astrocytes, and peripheral tissues. Proc. Natl. Acad. Sci. USA. 90: 4976-4980 [Abstract]. |
35. | Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex. EMBO (Eur. Mol. Biol. Organ.) J. 9: 61-67 [Abstract]. |
36. |
Maranto, A.R..
1994.
Primary structure, ligand binding, and localization of the human type 3 inositol 1,4,5-trisphosphate receptor
expressed in intestinal epithelium.
J. Biol. Chem.
269:
1222-1230
|
37. | Marchant, J.S., Y.-T. Chang, S.-K. Chung, R.F. Irvine, and C.W. Taylor. 1997. Rapid kinetic measurements of 45Ca2+ mobilization reveal that Ins(2,4,5)P3 is a partial agonist at hepatic InsP3 receptors. Biochem. J. 321: 573-576 [Medline]. |
38. |
Marks, A.R.,
P. Tempst,
C.C. Chadwick,
L. Riviere,
S. Fleischer, and
B. Nadal-Ginard.
1990.
Smooth muscle and brain inositol 1,4,5-trisphosphate receptors are structurally and functionally similar.
J. Biol. Chem.
265:
20719-20722
|
39. | Marshall, I., and C. Taylor. 1994. Two calcium binding sites mediate the interconversion of liver inositol 1,4,5-trisphosphate receptors between three conformations states. Biochem. J. 301: 591-598 [Medline]. |
40. | Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor. Nature (Lond.). 342: 192-195 [Medline]. |
41. | Mignery, G.A., and T.C. Sudhof. 1990. The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor. EMBO (Eur. Mol. Biol. Organ.) J. 9: 3893-3898 [Abstract]. |
42. |
Monkawa, T.,
A. Miyawaki,
T. Sugiyama,
H. Yoneshima,
M. Yamamoto-Hino,
T. Furuichi,
T. Saruta,
M. Hasegawa, and
K. Mikoshiba.
1995.
Heterotetrameric complex formation of inositol
1,4,5-trisphosphate receptor subunits.
J. Biol. Chem.
270:
14700-14704
|
43. | Morgan, J.M., J.I. Gillespie, and H. De Smedt. 1996. Identification of three isoforms of the InsP3 receptor in human myometrial smooth muscle. Pflügers Arch. 431: 697-705 [Medline]. |
44. |
Nathanson, M.H.,
M.B. Fallon,
P.J. Padfield, and
A.R. Maranto.
1994.
Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of the pancreatic acinar
cells.
J. Biol. Chem.
269:
4693-4696
|
45. | Negulescu, P.A., N. Shastri, and M.D. Cahalan. 1994. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc. Natl. Acad. Sci. USA. 91: 2873-2877 [Abstract]. |
46. |
Newton, C.,
G. Mignery, and
T. Sudhof.
1994.
Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3.
J. Biol.
Chem.
269:
28613-28619
|
47. | Nuccitelli, R., D.L. Yim, and T. Smart. 1993. The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins(1,4,5)P(3). Dev. Biol. 158: 200-212 [Medline]. |
48. | Othmer, H.G., and Y. Tang. 1993. Oscillations and waves in a model calcium dynamics. In Experimental and Theoretical Advances in Biological Pattern Formation. H.G. Othmer, J. Murray, and P. Maini, editors. Plenum Press, London. 295-319. |
49. | Petersen, C.C.H., E.C. Toescu, B.V.L. Potter, and O.H. Petersen. 1991. Inositol trisphosphate produces different patterns of cytoplasmic Ca2+ spiking depending on its concentration. FEBS Lett. 293: 179-182 [Medline]. |
50. |
Pietri, F.,
M. Hilly, and
J.-P. Mauger.
1990.
Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor.
J. Biol. Chem.
265:
17478-17485
|
51. | Putney, J.. 1990. The integration of receptor-regulated intracellular calcium release and calcium entry across the plasma membrane. Curr. Top. Cell. Regul. 31: 111-127 [Medline]. |
52. | Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature (Lond.). 339: 468-470 [Medline]. |
53. |
Somlyo, A.V.,
K. Horiuti,
D.R. Trentham,
T. Kitazawa, and
A.P. Somlyo.
1992.
Kinetics of Ca2+ release and contraction induced
by photolysis of caged D-myo-inositol 1,4,5-trisphosphate in
smooth muscle.
J. Biol. Chem.
267:
22316-22322
|
54. | Sudhof, T.C., C.L. Newton, B.T. Archer, Y.A. Ushkaryov, and G.A. Mignery. 1991. Structure of a novel InsP3 receptor. EMBO (Eur. Mol. Biol. Organ.) J. 10: 3199-3206 [Abstract]. |
55. | Sullivan, K.M.C., D.D. Lin, W. Agnew, and K.L. Wilson. 1995. Inhibition of nuclear vesicle fusion by antibodies that block activation of inositol 1,4,5-trisphosphate receptors. Proc. Natl. Acad. Sci. USA. 92: 8611-8615 [Abstract]. |
56. | Tang, Y., J.L. Stephenson, and H.G. Othmer. 1996. Simplification and analysis of models of calcium dynamics based on IP3-sensitive calcium channel kinetics. Biophys. J. 70: 246-263 [Abstract]. |
57. | Wakui, M., B.V.L. Potter, and O.H. Petersen. 1989. Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration. Nature (Lond.). 339: 317-320 [Medline]. |
58. | Walker, J.W., A.V. Somlyo, Y.E. Goldman, A.P. Somlyo, and D.R. Trentham. 1987. Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate. Nature (Lond.). 327: 249-252 [Medline]. |
59. |
Watras, J.,
I. Bezprozvanny, and
B.E. Ehrlich.
1991.
Inositol 1,4,5-trisphosphate-gated channels in cerebellum![]() |
60. | Watras, J., I. Moraru, D.J. Costa, and L.A. Kindman. 1994. Two inostiol 1,4,5-trisphosphate binding sites in rat basophilic leukemia cells: relationship between receptor occupancy and calcium release. Biochemistry. 33: 14359-14367 [Medline]. |
61. |
Wojcikiewicz, R.J.H..
1995.
Type I,II,III inositol 1,4,5-trisphosphate
receptors are unequally susceptible to down-regulation and are
expressed in markedly different proportions in different cell
types.
J. Biol. Chem.
270:
11678-11683
|
62. |
Worley, P.F.,
J.M. Baraban,
S. Supattapone,
V. Wilson, and
S.H. Snyder.
1987.
Characterization of inositol trisphosphate receptor
binding in brain.
J. Biol. Chem.
262:
12132-12136
|