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J. Biol. Chem., Vol. 281, Issue 13, 8332-8338, March 31, 2006

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Endogenously Bound Calmodulin Is Essential for the Function of the Inositol 1,4,5-Trisphosphate Receptor^*

Nael Nadif Kasri¹, Katalin Török, Antony Galione^¶, Clive Garnham^¶, Geert Callewaert, Ludwig Missiaen, Jan B. Parys, and Humbert De Smedt²

From the Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, O/N Herestraat 49/802, B-3000 Leuven, Belgium, ^¶Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom, and Department of Basic Medical Sciences, St. George's University of London, Cranmer Terrace, London SW17 0RE, United Kingdom

Received for publication, October 17, 2005 , and in revised form, December 19, 2005.

	ABSTRACT

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Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays an important role in regulating a large number of Ca²⁺ channels, including the inositol 1,4,5-trisphosphate receptor (IP₃R). Despite many efforts, the exact mechanism by which CaM regulates the IP₃R still remains elusive. Here we show, using unidirectional ⁴⁵Ca²⁺ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP₃R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP₃-induced Ca²⁺ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP₃. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP₃-induced Ca²⁺ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM₁₂₃₄, reactivated the IP₃R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP₃R, and this resulted in a complete loss of the IP₃R activity. Our data support a new model whereby CaM is constitutively associated with the IP₃R and functions as an essential subunit for proper functioning of the IP₃R.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The inositol 1,4,5 trisphosphate receptor (IP₃R)³ is a homo- or heterotetrameric intracellular Ca²⁺ release channel with a monomeric molecular mass of 310 kDa. In mammalian tissues, three isoforms have been identified (IP₃R1–3). All three isoforms are structurally and functionally related (1). The major part of the protein resides in the cytosol, where binding sites for IP₃ and various modulators (reviewed in Ref. 2) are located.

IP₃Rs are regulated in a complex way by Ca²⁺. Low Ca²⁺ concentrations ([Ca²⁺]) stimulate IP₃R activity, whereas higher [Ca²⁺] inhibits IP₃R activity (3–5). The positive regulation of the IP₃R by Ca²⁺ may be largely due to a direct binding of Ca²⁺ to the receptor (6, 7). The mechanism by which Ca²⁺ inactivates IP₃Rs is less clear. In that respect, much attention has already been focused on elucidating the role of calmodulin (CaM) (8).

CaM is a ubiquitous Ca²⁺-binding protein that plays an important role in Ca²⁺ signaling in many cell types by modulating the activity of numerous proteins, including ion channels (9). Each IP₃R subunit binds at least one CaM regardless of the [Ca²⁺] (10, 11). Both, Ca²⁺-dependent and -independent CaM-binding sites have been mapped to different regions of the IP₃Rs. A Ca²⁺-dependent CaM-binding site was identified in the regulatory domains of IP₃R1 and IP₃R2 (amino acids (aa) 1564–1595) (12), and a Ca²⁺-independent CaM-binding site was localized in the N-terminal parts of IP₃R1–3 (aa 49–81, aa 106–121) (13). At nanomolar [Ca²⁺], CaM does not affect IP₃R function, whereas at micromolar [Ca²⁺] CaM inhibits IP₃R function. The original hypothesis therefore was that Ca²⁺CaM mediates the Ca²⁺-dependent inactivation of IP₃Rs (14–16). However, this hypothesis became controversial, because we have recently shown, using a CaM mutant (CaM₁₂₃₄), that CaM is not a Ca²⁺ sensor, as such, for the IP₃R. We demonstrated that Ca²⁺-independent CaM binding to the N-terminal CaM-binding site is responsible for the CaM inhibition of IP₃-induced Ca²⁺ release (IICR), even though this inhibition requires Ca²⁺ (17). This can be explained by the large conformational change that the IP₃R undergoes in the presence of Ca²⁺ and which may be necessary for CaM action. This interaction may provide a tonic regulation of IP₃R activity and can explain the low sensitivity of the IP₃R in neuronal tissues where CaM is highly expressed. The role of the Ca²⁺-dependent CaM-binding site in the regulatory domain, however, still remains to be elucidated.

CaM-binding sites have not merely been identified as regulatory sites but have also recently been implicated in inter- and intrasubunit interactions. For example, the CaM-binding domain of the RyR1 modulates channel activity by at least two mechanisms: 1) by direct binding of CaM and 2) by forming a bridge between two different regions on the RyR1. Peptides of the RyR1 were used to demonstrate that the CaM-binding region is indeed directly involved in intersubunit interactions between the RyR subunits (18). In the case of small conductance K⁺ channels, CaM is involved in intersubunit interactions. Functional small conductance K⁺ channels are heteromeric complexes with CaM, which is constitutively associated with the -subunits in a Ca²⁺-independent manner (19). In this study, we investigated whether endogenously bound CaM is essential for IP₃R functioning. To test this hypothesis, we measured IP₃R activity in the presence of different synthetic peptides corresponding to the CaM-binding region of myosin light-chain kinase (MLCK), which has a very high affinity for CaM, and in the presence of peptides corresponding to CaM-binding sites of the IP₃R and other channels. We found that removing endogenously bound CaM dramatically reduced the efficacy of the IP₃R to release Ca²⁺. Our data support a new model whereby CaM is constitutively associated with the IP₃Ras an essential subunit for proper functioning of the IP₃R.

	MATERIALS AND METHODS

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Synthetic Peptides—The synthetic peptides derived from myosin light-chain kinase (MLCK, Ac-RRKWQKTGHAVRAIGRL-NH₂; and MLCK control peptide, Ac-RRKEQKTGHAVRAIGRE-NH₂) were obtained from Calbiochem. Peptides derived from IP₃R1 (peptide B, PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN; peptide E, ENRKLLGTVIQYGNVIQLLHLKS; peptide 1564–1595, KSHNIVQKTALNWRLSARNAARRDSVLAASRD), RyR1 (KSKKAVWHKLLSKQRRRAVVACFRMTPLYN), and hTrp3 (SFNSILNQPTRTQQIMKRLIKRYVLKAQVD) were all obtained from Sigma. Autocamtide 2-related inhibitory peptide (AIP) was obtained from Sigma.

⁴⁵Ca²⁺ Fluxes—L15 cells were obtained by stable exogenous expression of IP₃R1 in Lvec cells, whereas Lvec cells represent the control cells expressing the empty vector (20, 21). In some experiments, COS-1 cells were used. ⁴⁵Ca²⁺ fluxes were performed on saponin-permeabilized cells. The cells were seeded in 12-well clusters (Costar, MA) at a density of 4 x 10⁴ cm^–2. Experiments were carried out on confluent monolayers of cells (3 x 10⁵ cells/well) between the seventh and ninth day after plating. The cells were permeabilized by incubation for 10 min with a solution containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 2 mM MgCl₂, 1 mM ATP, 1 mM EGTA, and 20 µgml^–1 saponin at 25 °C. The non-mitochondrial Ca²⁺ stores were loaded for 45 min at 25 °C 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 ⁴⁵Ca²⁺ (28 µCi ml^–1). The cells were then washed twice with 1 ml of efflux medium containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 1 mM EGTA, and 4 µM thapsigargin. Thapsigargin was added to block the endoplasmic reticulum Ca²⁺ pumps during subsequent additions of Ca²⁺. The efflux medium was replaced every 2 min, and the efflux was performed at 25 °C. The additions of Ca²⁺, IP₃ and peptides are as indicated in the figures. Free [Ca²⁺] was calculated by the Cabuf program (ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) and based on the stability constants as previously published (22). At the end of the experiment, the ⁴⁵Ca²⁺ remaining in the stores was released by incubation with 1 ml of a 2% sodium dodecyl sulfate solution for 30 min. Ca²⁺ release in some experiments was plotted as fractional loss, i.e. the amount of Ca²⁺ released in 2 min divided by the total Ca²⁺ content at that time. The latter value was calculated by summing, in retrograde order, the amount of tracer remaining in the cells at the end of the efflux and the amounts of tracer collected during the successive time intervals.

Ca²⁺ Measurements in Sea Urchin Egg Homogenates—Agonist-induced calcium release was measured with the fluorescent dye fluo3 (3 µM) (Invitrogen) using Lytechinus pictus egg homogenates as previously described (23). Briefly, homogenates were diluted successively to 2.5% final v/v over 4 h at 17°C in Glu IM (250 mM potassium gluconate, 20 mM HEPES, and 1 mM MgCl₂ (pH 7.2)) supplemented by an ATP regeneration system (1 mM ATP, 10 units/ml creatine kinase, and 10 mM phosphocreatine) together with protease inhibitors. Aliquots of 100 µl of homogenate were added to each well in 96-well plates (Greiner BioOne) and read by a NOVOstar microplate reader (BMG Labtech, Aylesbury, UK). Additions of agonists were made in 1-µl volumes. Measurements were carried out at room temperature. Values were considered statistically different when p =< 0.05.

[³H]IP₃ Binding—Binding studies were performed on microsomes of IP₃R1-expressing Sf9 cells (150 µg) or microsomes derived from cerebellum (150 µg). [³H]IP₃ binding was performed at 0 °C in 160 µl of binding buffer containing 50 mM Tris-HCl (pH 7.0), 1 µM free [Ca²⁺], 10 mM -mercaptoethanol, and 10 nM [³H]IP₃. Nonspecific binding was determined in the presence of 12.5 µM unlabeled IP₃. After 30 min of incubation, the samples were rapidly filtered through glass fiber filters. Statistical analysis was performed using the paired Student's t test. Values were considered significantly different when p =<0.05.

Expression and Purification of CaM, CaM₁₂₃₄, CaM₁₂, CaM₃₄, and CaBP1—Proteins were expressed and purified as described previously (17, 24).

	RESULTS

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CaM-binding Peptide Derived from MLCK Decreases the IP₃R Activity—A synthetic CaM-binding peptide (Ac-RRKWQKTGHAVRAIGRL-NH₂) derived from the aa sequence of smooth muscle MLCK was previously characterized as an inhibitor of CaM activity. We used this peptide as a very effective inhibitor of CaM function, as the K_d for CaM was measured to be very low (6 pM) (25).

The non-mitochondrial Ca²⁺ stores of permeabilized L15 cells were first loaded to equilibrium with ⁴⁵Ca²⁺ and then incubated in efflux medium containing 0.5 µM free Ca²⁺. Thapsigargin (4 µM) was added to the efflux medium to allow a unidirectional Ca²⁺ efflux. Fig. 1A, filled squares, illustrates that a 2-min exposure to 1 µM IP₃ accelerated the rate of Ca²⁺ loss. At this concentration, IP₃ released 60 ± 4% of the maximum releasable Ca²⁺ measured by the addition of 5 µM A23187 [GenBank] . The IICR was much less pronounced when, prior to exposure with 1 µM IP₃, 10 µM MLCK peptide was added to the efflux medium for a period of 2 min. In this condition, 1 µM IP₃ released only 28 ± 5% of the maximum releasable Ca²⁺ (Fig. 1A, triangles).

Ca²⁺ release was always measured in the absence of Mg²⁺-ATP. Moreover, because there were four wash steps between the loading of the stores in the presence of Mg²⁺-ATP and the challenge with IP₃, all residual Mg²⁺-ATP should have been effectively removed. The involvement of the Ca²⁺- and CaM-dependent protein kinase (CaMKII) in the observed inhibition by the MLCK peptide seems therefore unlikely. In addition, we have also tested the effect of the CaMKII inhibitor AIP. KN93, which is also a common inhibitor, was not used because of its side effects on IICR (26). AIP had no effect on IICR, and the inhibition of IICR by the MLCK peptide (10 µM) was not affected (Fig. 1D). Similar data were observed with KN62, another CaMKII inhibitor (data not shown). We also used FK506 to exclude the role of calcineurin. Similar to AIP, FK506 did not show any effect on IICR (Fig. 1D). These findings exclude the involvement of two important CaM-binding proteins, CaMKII and calcineurin, in the inhibition of the IP₃R by the MLCK peptide.

Removal of Endogenously Bound CaM Causes Inhibition of IICR—It is conceivable that the observed decrease in IP₃R activity caused by the MLCK peptide might be due to either 1) a direct effect of the peptide on the IP₃R, 2) an indirect effect produced by the MLCK peptide binding endogenous CaM and thereby removing CaM from the IP₃R, or 3) a combination of the two effects. In an effort to distinguish between these possibilities, we have used a mutant of the MLCK peptide (MLCK control) in which a critical tryptophan and a leucine residue were mutated to a glutamate (Ac-RRKEQKTGHAVRAIGRE-NH₂). These mutations strongly reduced the affinity of the peptide for CaM from 6 pM to 10 µM (25). Application of this mutant peptide did not result in a decreased IICR (Fig. 1B). We used the peptide in conditions in which it does not bind CaM. As the MLCK peptide only binds Ca²⁺CaM, we measured IICR on permeabilized cells in efflux buffer containing 1 mM EGTA (Fig. 1C). In both cases, using MLCK peptide (Fig. 1C, triangles) or the MLCK-control peptide (circles) in the absence of Ca²⁺, no decrease in IICR was observed. These findings suggest that the inhibitory action of the MLCK peptide is dependent on its association with CaM.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. MLCK peptide inhibits IICR. A and B, after loading of permeabilized L15 cells during 45 min in 150 nM 45Ca2+, efflux was started in medium containing 0.5 µM free Ca2+. After stabilization of the efflux, these cells were challenged for 2 min with 1 µM IP3 (squares), as indicated by the arrow. Before challenging the cells with 1 µM IP3, cells were either incubated with 10 µM MLCK peptide (A, triangles) or 10 µM MLCK control peptide (B, triangles) for 2 min, as indicated by the dark bar. C, after loading of permeabilized L15 cells during 45 min in 150 nM 45Ca2+, efflux was started in Ca2+-free medium (1 mM EGTA). After stabilization of the efflux, the cells were challenged for 2 min with 1 µM IP3 (squares), as indicated by the arrow. Before challenging the cells with 1 µM IP3, cells were also incubated with 10 µM MLCK peptide (triangles) or 10 µM MLCK control peptide (circles) for 2 min, as indicated by the dark bar. Fractional loss was measured as a function of time and is defined as the amount of 45Ca2+ released in 2 min divided by the total amount of 45Ca2+ stored at that moment. Each curve represents the means ± S.E. of three independent experiments, each performed twice. D, Ca2+ release in L15 cells was measured upon the addition of 1 µM IP3 in the absence (white bars) or presence (gray bars) of 5 µM MLCK peptide. CaMKII inhibitor AIP (620 nM) or calcineurin inhibitor FK506 (5 µM) were added during efflux to evaluate their effect on IICR.

Next, we measured the dose-response curve on permeabilized cells that were incubated for 4 (Fig. 2, circles) or 6 (Fig. 2, triangles) min with MLCK peptide, replacing the peptide-containing efflux solution every 2 min. We observed that IICR was dramatically decreased when the peptide was added for a longer time. The IC₅₀ value shifted to 4.6 ± 0.5 and 2.1 ± 0.7 µM, respectively, for the 4- and 6-min incubations. In the absence of IP₃, we observed no effect of the peptide on the rate of efflux or on the endoplasmic reticulum Ca²⁺ content, when the peptide was added for a longer period (Fig. 2, inset). Both the concentration and the time dependence indicate that MLCK peptide inhibits IICR by removing endogenously bound CaM from the IP₃R. The dissociation of CaM from a high affinity binding site could be the rate-dependent step in this process. From the observation that the dissociation takes several minutes, it can be expected that the affinity is in the nanomolar or subnanomolar range.

Removal of Endogenously Bound CaM Alters the Extent of IICR but Not the Affinity of the IP₃R for IP₃—Previously, we have shown that adding exogenous CaM to permeabilized cells inhibited IICR (14, 17). This inhibition is Ca²⁺- and IP₃-dependent. Fig. 3A shows a dose-response curve of the Ca²⁺ release as a function of [IP₃] in the absence or presence of 10 µM CaM. Indeed, in L15 cells, 10 µM CaM inhibited IICR by increasing the EC₅₀ value from 0.44 ± 0.04 to 0.97 ± 0.07 µM but did not modify the extent of Ca²⁺ release at high [IP₃]. This is in accordance with our previously obtained data in A7r5 cells (14). In contrast, the addition of MLCK peptide (1 or 10 µM) had no effect on the EC₅₀ value for Ca²⁺ release compared with the control condition. The IC₅₀ value was 0.48 ± 0.07 and 0.46 ± 0.05 µM for 1 and 10 µM MLCK peptide, respectively (Fig. 3B). MLCK peptide, however, clearly reduced the extent of IICR. The addition of 10 µM MLCK peptide reduced the maximum IICR induced by 100 µM IP₃ from 84 ± 5 to 25 ± 6% of the total releasable Ca²⁺. The addition of exogenous CaM (10 µM) to 1 µM MLCK peptide prevented the above effects (data not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Time and concentration dependence of MLCK peptide inhibition of IICR. Ca2+ release from permeabilized L15 cells loaded with 45Ca2+ during 45 min was measured after stimulation with 1 µM IP3 in the presence of 0.5 µM Ca2+. Cells were incubated with increasing concentrations of MLCK peptide added for 2 (squares), 4 (circles), or 6 min (triangles). Ca2+ release was always plotted as the percentage of the maximum releasable fraction by 5 µM A23187 [GenBank] . Inset, Ca2+ stores were loaded for 45 min with 150 nM free 45Ca2+ and, from time 0 onward, incubated in efflux medium containing 0.5 µM Ca2+. The traces illustrate how the 45Ca2+ content of the stores decreased during the efflux in the absence of IP3 (inverted triangles) and demonstrate how this Ca2+ content was not affected by a 2- (squares), 4- (circles), or 6-min (triangles) application of 10 µM MLCK peptide after 2 min, as indicated by the bars. Results represent the means ± S.E. of three independent experiments, each performed twice.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. MLCK peptide alters the extent of IICR but not the affinity of the IP3R for IP3. A, Ca2+ release in L15 cells was measured as a function of IP3 in the absence (squares) or presence (circles) of 10 µM CaM. B, Ca2+ release in L15 cells was measured as a function of [IP3] in the absence (squares) or presence (triangles) of 1 µM or 10 µM MLCK peptide (circles) added for 6 min. Ca2+ release was always plotted as the percentage of the maximum releasable fraction by 5 µM A23187 [GenBank] . Results represent the means ± S.E. of three independent experiments, each performed twice. C, effect of 10 µM MLCK and MLCK control peptide on [3H]IP3 binding to Sf9 microsomes expressing IP3R1 (white bars) and microsomes derived from cerebellum (gray bars). Binding was measured at pH 7.0 in the presence of 1 µM free [Ca2+] and 10 nM [3H]IP3 and expressed as the percentage of the value in the absence of peptide.

Thus, the major effect of removing endogenously bound CaM by adding MLCK peptide appears to be a reduction in the extent of IICR rather than reducing the affinity of IP₃ for the IP₃R. This is in contrast to the effect of adding exogenous CaM to cells that were not exposed to MLCK peptide, where the major effect consists in reducing the affinity of the IP₃R for IP₃, without altering the extent of IICR (17).

Different CaM-binding Peptides Inhibit IICR—To confirm the idea that trapping endogenously bound CaM inhibits IICR, we reasoned that, by using other CaM-binding peptides, we should obtain similar results as those for MLCK peptide. Several CaM-binding peptides with different apparent affinities for CaM were used in a ⁴⁵Ca²⁺ flux assay. Permeabilized L15 cells were incubated for 2 min with 10 µM peptides derived from the RyR1 (aa 3614–3643; K_d, 10 nM) (27) and human homologue of Drosophila Trp (transient receptor potential) hTrp3 (aa 764–793; K_d, 100 nM) (28) prior to activation with 1 µM IP₃. We also used the CaM-binding peptides derived from the IP₃R1: peptide B (aa 49–81; K_d, 100 nM), peptide E (aa 106–128; K_d, 1 µM) (13), peptide 1564–1595 (aa 1564–1595; K_d, 64 nM) (12, 17). When the apparent affinity of the peptides was plotted as a function of the extent of inhibition after application of 1 µM IP₃ in the presence of 0.5 µM Ca²⁺, we observed a good correlation between the affinity of the peptide for CaM and the percentage of inhibition (Fig. 4). In the absence of Ca²⁺ in the efflux medium, RyR1 peptide and peptide E, which can bind Ca²⁺-free CaM, showed an inhibition of IICR of 22 ± 4 and 11 ± 6%, respectively, which is comparable with the inhibition in the presence of Ca²⁺ (Fig. 4). Other peptides that do not bind CaM in the absence of Ca²⁺ did not inhibit IICR (data not shown).

Effect of CaM-binding Peptides on Different IP₃R Isoforms—We measured the effects of MLCK peptide in different cell lines to evaluate the effects on the different IP₃R isoforms. The effect of 10 µM MLCK peptide was measured on permeabilized Lvec cells (expressing primarily IP₃R3) (see "Materials and Methods") and in COS-1 cells (expressing both IP₃R2 (25%) and IP₃R-3 (65%)). In both cell lines, the addition of 10 µM MLCK for 2 min inhibited IICR with 51 ± 3 and 40 ± 4%, respectively (data not shown). Although the effects were clearly present in three cell lines expressing different isoforms, it is difficult to conclude whether both isoforms present in COS-1 cells equally contributed to the observed inhibition. From the data in L15 and Lvec cells, we can, however, conclude that IICR is effectively inhibited for at least IP₃R1 and -3.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Inhibition of IICR by CaM-binding peptides. Ca2+ release induced by 1 µM IP3 was measured in the presence of 0.5 µM Ca2+ and 10 µM of various CaM-binding peptides added for 2 min prior to challenging the cells with IP3. CaM-binding peptides used were derived from MLCK, RyR1, hTrp3, and IP3R1. The percentage of inhibition on IICR versus the control condition is plotted as a function of the published Kd value of the peptides for CaM. Results represent the means ± S.E. of three independent experiments, each performed twice.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Effect of CaM-binding peptides on sea urchin egg homogenates. A and B, sea urchin egg homogenates were loaded with 3 µM fluo3 50 min prior to stimulation with 2 µM IP3 (A) or 200 nM cADPR (B); homogenates were incubated with 50 µM MLCK or MLCK control peptide. The data represent the rise in fluorescence as a percentage of the control condition and are represented as the means ± S.E. Statistical significance is indicated by *, p < 0.05.

Reversing the Effects of the Peptide by Re-addition of CaM—We assessed the possibility of reversing the inhibition on IICR caused by the removal of CaM by re-adding exogenous CaM. We showed earlier that adding exogenous CaM inhibited IICR (Fig. 3A) in an IP₃-dependent way. Therefore we chose to perform the experiment at an [IP₃] of 100 µM. At this high [IP₃], exogenous CaM did not inhibit IICR, whereas IICR was still largely inhibited by MLCK peptide (Fig. 3). Permeabilized L15 cells were first incubated with 1 µM peptide for 6 min, and efflux medium with peptide was then replaced by efflux medium with (Fig. 6, triangles) or without (Fig. 6, circles) 1 µM CaM for 4 min. The cells were then challenged with 100 µM IP₃, and IICR was compared with cells that were only challenged with 100 µM IP₃ (Fig. 6, squares). In the condition in which only MLCK peptide was added, IICR was inhibited by 42 ± 4% compared with the control condition. In the condition in which exogenous CaM was added after the addition of the peptide, IICR was only reduced by 14 ± 5%. This implicates that adding exogenous CaM after having removed CaM with MLCK-peptide partially restores the activity of the IP₃R. The same experiment was performed with a mutant CaM, which was mutated in the 4 EF-hands, CaM₁₂₃₄ (Fig. 6, inverted triangles). In contrast to CaM, CaM₁₂₃₄ did not restore IICR. We further investigated whether CaM mutants, mutated in the N-terminal EF-hands (CaM₁₂) or in the C-terminal EF-hands (CaM₃₄), could restore IICR. But neither CaM₁₂ nor CaM₃₄ restored IICR. A CaM-like protein, i.e. calcium-binding protein-1 (CaBP1), which was recently shown to interact with the N-terminal CaM-binding site of the IP₃R (24, 29–30), could also not restore IICR (data not shown). We conclude that only fully Ca²⁺-loaded Ca²⁺CaM is able to reverse the inhibition by MLCK peptide.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CaM is the first protein to have emerged as a potential inhibitory Ca²⁺ sensor for the IP₃R, but the exact nature of its involvement remains unclear. Exogenous CaM inhibits IICR in a Ca²⁺-dependent way in various cell types expressing different combinations of IP₃R isoforms (14, 16, 31). In a previous study, we have shown that the Ca²⁺-dependent inhibition occurs through binding of CaM to the N-terminal Ca²⁺-independent CaM-binding site. The Ca²⁺ dependence then was proposed to be a property of the IP₃R itself. We suggest that CaM is endogenously bound to the IP₃R (17).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Effect of exogenous CaM on inhibition by MLCK peptide. After loading of permeabilized L15 cells during 45 min in 150 nM 45Ca2+, efflux was started in medium containing 0.5 µM free Ca2+. After stabilization of the efflux, the cells were challenged for 2 min with 100 µM IP3 (squares), as indicated by the arrow. Before challenging the cells with IP3, cells were incubated with 10 µM MLCK peptide for 6 min (circles) and subsequently with 1 µM CaM (triangles) or 1 µM CaM1234 (inverted triangles), as indicated by the bars. Results represent the means ± S.E. of three independent experiments, each performed twice.

We therefore tested the hypothesis that CaM is endogenously bound to the IP₃R and might be involved in inter- or intrasubunit interactions by measuring the effects on IICR of different synthetic peptides representing CaM-binding regions. First, we used the CaM-binding region derived from MLCK, as this peptide was found to bind CaM with an extremely high affinity (K_d, 6 pM) (25). The results demonstrated that adding MLCK peptide to permeabilized L15 cells dramatically decreased IICR. We showed that MLCK peptide caused a reduction in the extent of IICR with no change in the affinity of IP₃ for the IP₃R. A direct effect of the different CaM-binding peptides on the IP₃R is unlikely, as except for their CaM-binding abilities, they have no particular homology. Furthermore, the observation that MLCK peptide was only effective in the presence of Ca²⁺, whereas the Ca²⁺-independent RyR peptide also inhibited IICR in the absence of Ca²⁺, is more compatible with the removal of endogenous CaM than with binding of these peptides directly to the IP₃R. Furthermore, using inhibitors for CaMKII and calcineurin, we also excluded the involvement of both CaM-binding proteins. Although we cannot fully exclude the effect of an as yet unknown auxiliary CaM-binding protein, we therefore concluded that removal of endogenous CaM from the IP₃R was the most probable cause of the inhibition of IICR. This hypothesis was strengthened by reversing the inhibitory effects of the peptides by adding exogenous CaM but not CaM₁₂₃₄, CaM₁₂, or CaM₃₄.

Previously, CaBP1 was identified as a novel activator of the IP₃R (29). Although this is still controversial (24, 30), we have envisaged the hypothesis that CaBP1 could activate IP₃R after complete removal of CaM from the IP₃R. However, we did not observe any activation by CaBP1 alone or potentiation of IICR after removal of the endogenously bound CaM. This indicates that the N-terminal CaM-binding site that is also used by CaBP1 (24) is not involved in this process.

Interestingly, exogenously added CaM and removal of endogenously bound CaM inhibited IICR by a completely different mechanism. Although adding exogenous CaM inhibited IICR by reducing the affinity of the IP₃R for IP₃, removing endogenously bound CaM with the peptides did not alter the IC₅₀ value for IP₃ but dramatically decreased the maximum release capacity of the IP₃R. This indicates that a different CaM-binding site than the N-terminal CaM-binding site must be involved. The CaM-binding site that was previously identified in the regulatory region of IP₃R1 and IP₃R2 (aa 1564–1585) can also not explain the effect observed here, as this site is not conserved in IP₃R3 (12). The inhibitory effects were observed in IP₃R1 and IP₃R3, because they were observed in different cell types, expressing mainly either one of these isoforms. These inhibitory effects were also observed in sea urchin egg homogenates. The effects probably therefore involve an as yet unidentified CaM-binding site on the IP₃R.

Recently, major progress has been made in understanding the gating mechanisms of the IP₃R (37). It becomes clear from the data that intramolecular interactions control the opening and closing of the channel. Reports demonstrated the proximity and physical interaction of the ligand-binding domain with the IP₃R channel domain (38, 39). The truncated receptor containing only the transmembrane segments, i.e. the channel domain, was shown to be constitutively active (40). In addition, studies by Ramos-Franco et al. (41) show that a truncated IP₃R missing transmembrane segments 1–4 also forms a constitutively open channel. Different regions may therefore contain important domains to keep the channel closed. Current views suggest that IP₃ binding initiates a conformational change or perhaps a series of conformational changes that relieve the inhibitory effect of a yet unidentified segment of the regulatory region (42) and/or the transmembrane segments 1–4 (41). One could speculate that CaM is directly involved in stabilizing these intramolecular interactions or that CaM is directly involved in the gating mechanism itself. Removing this endogenous CaM would disable the IP₃R to open or close properly. As the endoplasmic reticulum content is not affected by the CaM-binding peptides, we suggest that the removal of CaM stabilizes the IP₃R in a closed conformation. In the case of the RyR1, disrupting the intersubunit interactions directly altered the opening properties of the channel, leading to an increase in spontaneous Ca²⁺ sparks in frog skeletal muscle. The CaM-binding site in the RyR1 is thought to be at an intersubunit contact site, and depletion of CaM may sensitize the channel to activation by disrupting this intersubunit interaction (18, 32).

Our data are most easily interpreted as depletion of endogenous CaM, because the effect is proportional to the affinity of the peptide for CaM. This depletion did not empty the internal Ca²⁺ stores, indicating that the IP₃R remained in a closed state. CaM seems to be required for the coupling mechanism between IP₃ binding and channel opening, perhaps by stabilizing the IP₃-induced conformational change.

In summary, we have shown that a high affinity CaM-binding peptide strongly decreased the ability of the IP₃R to release Ca²⁺. We found that stripping CaM from the IP₃R was responsible for this inhibition. These results are consistent with a model in which CaM is not merely a regulator of the IP₃R but is also an essential component in the proper gating of the IP₃R.

	FOOTNOTES

 
^* This work was supported by Grants G.0210.03 (to H. D. S. and J. B. P.) G.O0382.05 (to L. M. and G. C.) of the Fund for Scientific Research Flanders (Belgium), by Grant GOA2004/07 from the Concerted Actions of the K. U. Leuven Institute (to L. M., H. D. S., G. C., and J. B. P.) and by the Interuniversity Poles of Attraction Programme— Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs, IUAP P5/05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A postdoctoral fellow of the Research Foundation (FWO-Vlaanderen) Brussels, Belgium.

² To whom correspondence should be addressed: Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Herestraat 49/802, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-25; Fax: 32-16-34-59-91; E-mail: humbert.desmedt{at}med.kuleuven.be.

³ The abbreviations used are: IP₃R, IP₃ receptor; CaBP1, calcium-binding protein-1; CaM, calmodulin; IP₃, inositol 1,4,5-trisphosphate; IICR, IP₃-induced Ca²⁺ release; MLCK, myosin light-chain kinase; aa, amino acids; AIP, autocamtide 2-related inhibitory peptide; CaMKII, Ca²⁺- and CaM-dependent protein kinase.

	ACKNOWLEDGMENTS

 
We thank Lea Bauwens, Marina Crabbé, Anja Florizoone, Sylvie De Swaef, Silvia Vangeel, and Tomas Luyten for technical assistance. The mammalian CaM cDNA was kindly provided by Dr. Z. Grabarek (Boston Biomedical Research Institute, Boston, MA) and the rat cDNA for CaM₁₂, CaM₃₄, and CaM₁₂₃₄ were kindly provided by Dr. J. Adelman (Vollum Institute, Portland, OR).

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