COMMUNICATION
The Bell-shaped Ca2+ Dependence of the Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Is Modulated by Ca2+/Calmodulin*

Ludwig MissiaenDagger , Jan B. Parys§, A. Freek Weidema, Henk Sipma, Sara Vanlingen, Patrick De Smet, Geert Callewaert, and Humbert De Smedt

From the Laboratorium voor Fysiologie, Katholieke Universiteit Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 µM if the cytosolic Ca2+ concentration was 0.3 µM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 µM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 µM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Inositol 1,4,5-trisphosphate (IP3)1 is a second messenger used by many cell types to release Ca2+ from internal stores (1). Cytosolic Ca2+ has a bell-shaped effect on the IP3 receptor (IP3R), with low concentrations stimulating the release and high concentrations inhibiting it (2-5). Calmodulin, a Ca2+-binding protein abundantly present in many cell types (6), binds to the modulatory region of the IP3R in a Ca2+-dependent way (7, 8). Calmodulin also interacts with the IP3R in a Ca2+-independent way (9-11), with one of the interaction sites located within the IP3-binding domain (11). The findings that calmodulin inhibited IP3-induced Ca2+ release in a medium containing 0.2 µM free Ca2+ and in addition inhibited [3H]IP3 binding both in the absence and presence of cytosolic Ca2+ led to the proposal that the Ca2+-independent binding of calmodulin was responsible for the regulation of the IP3R (9).

Although the free cytosolic [Ca2+] is a very important regulator of the IP3R (2-5), there are no functional data showing how calmodulin modulates the Ca2+ release induced by IP3 at various Ca2+ concentrations. We have therefore studied how Ca2+ interferes with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. All experiments were performed in the absence of Mg-ATP to avoid activation of the Ca2+- and calmodulin-dependent protein kinase CaMKII that was reported to stimulate the IP3R (12). We now report that calmodulin inhibited the IP3-induced Ca2+ release if the free cytosolic [Ca2+] was 0.3 µM or higher. This inhibition occurred with an IC50 of 4.6 µM and was particularly pronounced at low IP3 concentrations. Calmodulin did not affect the IP3-induced Ca2+ release at lower Ca2+ concentrations. The effects of Ca2+ could be mimicked by Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release. As a consequence, the bell-shaped Ca2+ activation curve of the IP3R becomes narrower in the presence of calmodulin.

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45Ca2+ fluxes were performed on saponin-permeabilized A7r5 cells from embryonic rat aorta (13). The nonmitochondrial Ca2+ stores were loaded for 50 min in 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN3, and 150 nM free Ca2+ (23 µCi ml-1) and then washed once in 1 ml of efflux medium containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 4 µM thapsigargin, and, unless otherwise indicated, 1 mM BAPTA. The efflux medium was replaced every 2 min for 20 min. The additions of IP3, Ca2+, Sr2+, and calmodulin are indicated in the figures. The free [Ca2+] was calculated with the CaBuf computer program using the following decimal logarithms of the association constants for ATP: H-ATP, 6.49; H-HATP, 4.11; Ca-ATP, 3.78; Ca-HATP, 1.98; Mg-ATP, 4.00; and Mg-HATP, 2.06 (14). The association constants for BAPTA were: H-BAPTA, 6.36; H-HBAPTA, 5.47; Ca-BAPTA, 6.97; and Sr-BAPTA, 5.13 (15). At the end of the experiment the 45Ca2+ remaining in the stores was released by incubation with 1 ml of a 2% SDS solution for 30 min.

Calmodulin from bovine brain (purity >99%) was obtained from Calbiochem (San Diego, CA) and dissolved as a 1 mM stock in 30 mM imidazole-HCl (pH 6.8). Control samples were treated with the same buffer.

    RESULTS AND DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Effect of Calmodulin on IP3-induced Ca2+ Release in the Presence of 0.3 µM Free Ca2+-- Permeabilized A7r5 cells loaded to equilibrium with 45Ca2+ slowly lost their accumulated 45Ca2+ during incubation in efflux medium containing 1 mM BAPTA and no added Ca2+. Thapsigargin (4 µM) was added to block the endoplasmic reticulum Ca2+ pumps during the additions of Ca2+. A short exposure to 1 µM IP3 and 0.3 µM free Ca2+ accelerated the rate of Ca2+ loss (Fig. 1a, closed circles). The release was less pronounced if 10 µM calmodulin was added together with the IP3 and Ca2+ (Fig. 1a, open circles). Addition of 0.3 µM free Ca2+ in the absence of IP3 by itself induced a small Ca2+ release (Fig. 1b), due to the exchange of labeled Ca2+ for unlabeled Ca2+ (3, 16). This release was identical in the presence (Fig. 1b, open circles) or absence (Fig. 1b, closed circles) of 10 µM calmodulin.


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Fig. 1.   Effect of calmodulin on the Ca2+ release induced by 1 µM IP3 and 0.3 µM free Ca2+ in permeabilized A7r5 cells. a, the nonmitochondrial Ca2+ stores were loaded to steady state with 45Ca2+ and then incubated in efflux medium containing 1 mM BAPTA and no added Ca2+. At the time indicated by the horizontal bar, 1 µM IP3 and 0.3 µM free Ca2+ were added for 2 min in the absence () or presence (open circle ) of 10 µM calmodulin. The stores in b were treated with 0.3 µM Ca2+ in the absence () or presence (open circle ) of 10 µM calmodulin, as indicated by the bar. Ca2+ release is plotted as fractional loss, i.e. the amount of Ca2+ released in 2 min divided by the total store Ca2+ content at that time. The means of five experiments are shown. The S.E. was always less than 5%.

Ca2+ release was always measured in the absence of Mg-ATP. Moreover, because there were six wash steps between the loading of the stores in the presence of Mg-ATP and the challenge with IP3, all residual Mg-ATP should have been effectively removed. The involvement of the Ca2+- and calmodulin-dependent protein kinase CaMKII in the observed inhibition by calmodulin seems therefore unlikely. In addition, we have also tested the effect of the CaMKII inhibitor KN62. Calmodulin (10 µM) inhibited the Ca2+ release induced by 1 µM IP3 and 0.3 µM free Ca2+ by 54 ± 3% in the absence of KN62 and by 51 ± 4% (n = 3) in the presence of 10 µM KN62. These findings exclude the involvement of CaMKII in the inhibition of the IP3R by calmodulin.

[Ca2+] Dependence of the Effect of Calmodulin on IP3-induced Ca2+ Release-- Similar experiments to those illustrated in Fig. 1 were performed at several free Ca2+ concentrations (Fig. 2a). Calmodulin (10 µM) did not inhibit the IP3R in the absence of added Ca2+ or in the presence of low free Ca2+ concentrations (0.03 or 0.1 µM). The same concentration of calmodulin, however, strongly inhibited the IP3R at higher free Ca2+ concentrations (0.3 and 1 µM).


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Fig. 2.   The effect of calmodulin on the IP3R in permeabilized A7r5 cells is [Ca2+]-dependent. The IP3-induced Ca2+ release in efflux medium containing 1 mM (a) or 6 mM (b) BAPTA was calculated as the difference between the Ca2+ release in the presence and that in the absence of IP3 (3). The free [Ca2+] during the IP3 application is indicated on the abscissa. The closed bars represent the Ca2+ release induced by 1 µM IP3 in the absence of calmodulin. o and oo, significantly different from the Ca2+ release in the absence of added Ca2+ with p < 0.01 and p < 0.05, respectively, as determined using Student's t test for paired samples. The hatched bars represent the Ca2+ release in the presence of 10 µM calmodulin. *, significantly different from the control at the same [Ca2+] in the absence of calmodulin (p < 0.01) as determined using Student's t test for paired samples. Means ± S.E. are shown for five experiments, each performed in duplicate.

Because the calmodulin used was lyophilized from a dialysis buffer containing 30 µM Ca2+, four Ca2+ ions were bound to each molecule of calmodulin. We calculated that the addition of 10 µM calmodulin and its contaminating 40 µM Ca2+ to the efflux medium containing 1 mM BAPTA and 650 µM total Ca2+ could therefore have slightly increased the free [Ca2+] from 0.30 to 0.36 µM. Although unlikely, we wanted to exclude that the inhibitory effect of calmodulin could be caused by this small change in free [Ca2+]. Addition of 40 µM Ca2+ in the absence of calmodulin inhibited the Ca2+ release induced by 1 µM IP3 and 0.3 µM free Ca2+ by only 4.3 ± 2.2% (n = 3). We have also tested the effect of calmodulin in an efflux medium containing 6 mM BAPTA instead of 1 mM BAPTA. The addition of 10 µM calmodulin to efflux medium containing 6 mM BAPTA and 3.897 mM total Ca2+ increased the calculated free [Ca2+] from 0.30 to only 0.31 µM. Fig. 2b shows that under these conditions of strong Ca2+ buffering, 10 µM calmodulin still inhibited the IP3R in the presence of 0.3 and 0.6 µM free Ca2+. The same concentration of calmodulin had again no effect on the IP3-induced Ca2+ release at lower free Ca2+ concentrations. Fig. 2 also illustrates that cytosolic Ca2+ exerted its biphasic effect on the IP3R both in the presence and in the absence of calmodulin. In the presence of calmodulin, the inhibition occurred at lower Ca2+ concentrations. As a consequence, the bell-shaped Ca2+ activation curve of the IP3R became narrower in the presence of calmodulin.

The [Ca2+]-dependence of the IP3-induced Ca2+ release was markedly different when Ca2+ was buffered with 1 mM (Fig. 2a) or 6 mM BAPTA (Fig. 2b). Both the stimulatory and inhibitory effects of Ca2+ were more pronounced at the higher concentration of BAPTA. It is possible that this difference is caused by the postulated local [Ca2+] rise in the vicinity of the IP3Rs as a result of the passive Ca2+ leak from the stores (17, 18). This [Ca2+] rise will be less pronounced in the presence of 6 mM BAPTA, thereby reducing the IP3-induced Ca2+ release at the lowest Ca2+ concentration from 46.9 ± 2.1 to 30.3 ± 2.3%/2 min (n = 5). As a consequence the stimulatory effect of elevating the [Ca2+] was more pronounced in the presence of 6 mM BAPTA. An alternative possibility could be that the IP3R at low free Ca2+ concentrations is inhibited by the Ca2+-free form of BAPTA, which is the predominant form of the chelator under these conditions (19, 20). Such inhibition would be more pronounced at 6 mM BAPTA, which could again explain why the release in the absence of Ca2+ was reduced at the higher concentration of BAPTA. However, we have previously shown that this inhibitory effect was relatively small in A7r5 cells (21).

The Inhibition of the IP3R by Calmodulin Is Dose-dependent-- Fig. 3a illustrates the Ca2+ release induced by 1 µM IP3 and 0.3 µM free Ca2+ in the presence of various concentrations of calmodulin. Calmodulin inhibited the IP3R with an IC50 of 4.6 µM and a Hill-coefficient of 1.0, which is consistent with a single interaction with no evidence for cooperativity between the subunits of the IP3R tetramer.


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Fig. 3.   Inhibition of the IP3R by calmodulin in permeabilized A7r5 cells: effects of the [calmodulin] and [IP3]. a, Ca2+ release induced by 1 µM IP3 and 0.3 µM free Ca2+ in the presence of the indicated [calmodulin]. The Ca2+ release in the absence of calmodulin was taken as 100%. b, stores were challenged with the indicated [IP3] in the absence () or presence (open circle ) of 10 µM calmodulin in a medium containing 0.3 µM free Ca2+. Means ± S.E. are shown for four (a) and three (b) experiments, each performed in duplicate. *, significantly different from the control in the absence of calmodulin (p < 0.02) as determined using Student's t test for paired samples.

Inhibitory Effect of Calmodulin on the Ca2+ Release Induced by Various IP3 Concentrations-- Fig. 3b shows the Ca2+ release as a function of the [IP3] in the absence (closed circles) and presence (open circles) of 10 µM calmodulin in a medium containing 0.3 µM free Ca2+. IP3 stimulated the IP3R with an EC50 of 0.25 µM IP3 in the absence of calmodulin and with an EC50 of 2.9 µM IP3 in the presence of calmodulin. Calmodulin not only increased the EC50 for IP3-induced Ca2+ release but also decreased the maximal Ca2+ release induced by high IP3 concentrations. Interestingly, the inhibition was relatively more pronounced at lower IP3 concentrations, e.g. 10 µM calmodulin caused an 82% inhibition of the Ca2+ release induced by 0.3 µM IP3, whereas that in the presence of 300 µM IP3 was only inhibited by 20%.

[Sr2+] Dependence of the Effect of Calmodulin on the IP3-induced Ca2+ Release-- The inhibitory effects of calmodulin were clearly dependent on the presence of Ca2+. To discriminate whether calmodulin acted by potentiating the inhibitory effects of Ca2+ or whether the requirement for Ca2+ to see the inhibition by calmodulin reflected the formation of Ca2+/calmodulin, we have studied the effect of calmodulin in the presence of various Sr2+ concentrations. Sr2+ is only 3-fold less potent than Ca2+ in activating the liver IP3R but is 600-fold less potent in inhibiting it (22). A similar effect was observed in A7r5 cells, where Sr2+ up to 100 µM induced a concentration-dependent decrease in the EC50 for IP3-induced Ca2+ release, whereas Ca2+ induced a biphasic effect with low Ca2+ concentrations decreasing the EC50 and higher Ca2+ concentrations increasing it (23). Fig. 4 shows the Ca2+ release induced by 1 µM IP3 in the presence of increasing Sr2+ concentrations. The closed bars confirm that Sr2+ activated the IP3R and that no significant inhibition was observed at 30 µM Sr2+. The hatched bars show the effect of 10 µM calmodulin. Calmodulin did not inhibit the IP3R in the absence of added Sr2+ or in the presence of low free Sr2+ concentrations (1 or 3 µM). The same concentration of calmodulin, however, strongly inhibited the IP3R at higher free Sr2+ concentrations (10 and 30 µM), which by themselves were not inhibitory. Because Sr2+ binds to calmodulin (24) but does not inhibit the IP3R in the absence of calmodulin, we conclude that the dependence of the calmodulin inhibition on the presence of Sr2+ or Ca2+ represents the formation of Sr2+/calmodulin or Ca2+/calmodulin. The inhibition occurred at higher Sr2+ concentrations than Ca2+ concentrations, probably because Sr2+ was 30 times less effective than Ca2+ in binding to the high affinity Ca2+-binding sites of calmodulin (24).


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Fig. 4.   The effect of calmodulin on the IP3R in permeabilized A7r5 cells at various Sr2+ concentrations. The IP3-induced Ca2+ release in efflux medium containing 1 mM BAPTA was calculated as the difference between the Ca2+ release in the presence and that in the absence of IP3 (3). The free [Sr2+] during the IP3 application is indicated on the abscissa. The closed bars represent the Ca2+ release induced by 1 µM IP3 in the absence of calmodulin. o and oo, significantly different from the Ca2+ release in the absence of added Sr2+ with p < 0.01 and p < 0.05, respectively, as determined using Student's t test for paired samples. The hatched bars represent the Ca2+ release in the presence of 10 µM calmodulin. *, significantly different from the control at the same [Sr2+] in the absence of calmodulin (p < 0.02) as determined using Student's t test for paired samples. Means ± S.E. are shown for seven experiments.

Conclusions-- We observed that calmodulin inhibited the IP3-induced Ca2+ release if the free cytosolic [Ca2+] was 0.3 µM or higher, whereas there was no effect at lower free Ca2+ concentrations. Calmodulin therefore shifted the Ca2+-dependent inhibition of the IP3R toward lower free Ca2+ concentrations without affecting the Ca2+-dependent activation. This results in a narrower bell-shaped dependence of the IP3R on Ca2+, which may be important for inducing the termination of intracellular Ca2+ spikes. Because all experiments were done in the absence of Mg-ATP and because the inhibition was not affected by 10 µM KN62, the involvement of a Ca2+- and calmodulin-dependent protein kinase can be excluded. The IP3R interacts with calmodulin in a Ca2+-dependent (7-9) and a Ca2+-independent way (9-11). It has been proposed that the Ca2+-independent interaction was responsible for the inhibition of the release (9), because calmodulin inhibited IP3-induced Ca2+ release in the presence of 0.2 µM free Ca2+ and inhibited [3H]IP3 binding at all free Ca2+ concentrations tested. Our data on the effects of calmodulin on the Ca2+ release induced by IP3 and a broad range of cytosolic Ca2+ concentrations now indicate that the inhibition of the IP3-induced Ca2+ release is strictly dependent on the formation of a Ca2+/calmodulin or a Sr2+/calmodulin complex.

The results in the present study showing that the inhibitory effect of calmodulin on IP3-induced Ca2+ release was dependent upon cytosolic Ca2+ or Sr2+ are difficult to reconcile with previous studies demonstrating a Ca2+-independent inhibition of IP3 binding to purified cerebellar IP3Rs (9), cerebellar membranes (9), type-1 IP3Rs expressed in Sf9 cells (10, 11), and the recombinant IP3-binding domain of the type-1 IP3R (11). It was technically impossible to study the effects of calmodulin on [3H]IP3 binding to A7r5 microsomes. Indeed, despite repeated attempts at different ligand concentrations, the low density of IP3Rs in A7r5 cells precluded the detection of IP3 binding at neutral pH values, although it can be easily measured at alkaline pH (25). The measurements at neutral pH were necessary, because the reported Ca2+-independent effects of calmodulin on [3H]IP3 binding disappeared at alkaline pH, probably due to a conformational change of calmodulin (9, 11). The apparent discrepancy between the functional data reported in the present study and the [3H]IP3 binding data (9-11) may be due to the existence of multiple calmodulin-binding or Ca2+-binding sites that play a role and/or to the fact that IP3 binding, which is only one step, albeit a crucial step, for channel opening, is not equivalent to the channel activity itself.

    ACKNOWLEDGEMENT

We thank Dr. G. Droogmans (Laboratory of Physiology, Katholieke Universiteit Leuven) for the computer program CaBuf to measure the free [Ca2+] and [Sr2+].

    FOOTNOTES

* This work was supported by the Interuniversity Poles of Attraction Program, Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs IUAP P4/23 and by European Commission Grant BMH4-CT96-0656.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 32-16-345720; Fax: 32-16-345991; E-mail: Ludwig.Missiaen{at}med.kuleuven.ac.be.

§ Research Associate of the Foundation for Scientific Research-Flanders.

Senior Research Assistant of the Foundation for Scientific Research-Flanders.

    ABBREVIATIONS

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'tetraacetic acid.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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