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Originally published In Press as doi:10.1074/jbc.M602175200 on May 24, 2006

J. Biol. Chem., Vol. 281, Issue 30, 20825-20833, July 28, 2006
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CIB1, a Ubiquitously Expressed Ca2+-binding Protein Ligand of the InsP3 Receptor Ca2+ Release Channel*Formula

Carl White{ddagger}, Jun Yang{ddagger}, Mervyn J. Monteiro§, and J. Kevin Foskett{ddagger}1

From the Departments of {ddagger}Physiology and Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and §University of Maryland Biotechnology Institute and Medical Biotechnology Center, University of Maryland, Baltimore, Maryland 21201

Received for publication, March 7, 2006 , and in revised form, May 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A family of Ca2+-binding proteins (CaBPs) was shown to bind to the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and gate it in the absence of InsP3, establishing them as protein ligands (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711–7716). However, the neuronally restricted expression of CaBP and its inhibition of InsP3R-mediated Ca2+ signaling when overexpressed (Kasri, N. N., Holmes, A. M., Bultynck, G., Parys, J. B., Bootman, M. D., Rietdorf, K., Missiaen, L., McDonald, F., De Smedt, H., Conway, S. J., Holmes, A. B., Berridge, M. J., and Roderick, H. L. (2004) EMBO J. 23, 312–321; Haynes, L. P., Tepikin, A. V., and Burgoyne, R. D. (2004) J. Biol. Chem. 279, 547–555) have raised questions regarding the functional implications of this regulation. We have discovered the Ca2+-binding protein CIB1 (calmyrin) as a ubiquitously expressed ligand of the InsP3R. CIB1 binds to all mammalian InsP3R isoforms in a Ca2+-sensitive manner dependent on its two functional EF-hands and activates InsP3R channel gating in the absence of InsP3. In contrast, overexpression of CIB1 or CaBP1 attenuated InsP3R-dependent Ca2+ signaling, and in vitro pre-exposure to CIB1 reduced the number of channels available for subsequent stimulation by InsP3. These results establish CIB1 as a ubiquitously expressed activating and inhibiting protein ligand of the InsP3R.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium mobilization from the endoplasmic reticulum (ER)2 through the inositol 1,4,5-trisphopshate (InsP3) receptor (InsP3R) Ca2+ release channel is a ubiquitous signaling system that is central to the regulation of numerous cellular processes, ranging from transcription to synaptic plasticity (1). InsP3-mediated Ca2+ signals display complex spatial and temporal features that vary among cell types and have been attributed to the subcellular distribution and expression levels of three InsP3R genes with alternatively spliced isoforms (2–4) and complex regulation of the channel by its ligands InsP3 and Ca2+ (5–7), as well as modulation by ATP (8, 9), phosphorylation (10–12), and protein interactions (7).

The InsP3-liganded InsP3R is regulated by [Ca2+]i in a complex manner. Increases of [Ca2+]i from resting levels (50 nM) to ~1 µM stimulate channel gating (5, 13–15), enabling the channel to participate in Ca2+-induced Ca2+ release, which couples the activities of individual InsP3R within channel clusters and transforms local [Ca2+]i signals to global and propagating ones. High [Ca2+]i (>10 µM) achieved in close proximity to the mouths of Ca2+ channels inhibit InsP3R gating (5, 13, 15), which may play a role in both terminating Ca2+ release and preventing channel activation. Notably, InsP3 and ATP affect channel activity by allosteric modulation of the Ca2+ regulation of the channel (8, 9). Thus, the mechanisms underlying [Ca2+]i regulation of InsP3R activity are of central importance in determining the properties of [Ca2+]i signals in cells. Nevertheless, the molecular details of this regulation are still unknown. The relative roles of Ca2+ binding to the InsP3R and indirect regulation by Ca2+ binding to other proteins have been debated. Thus, it has been suggested that high [Ca2+]i inhibition of the InsP3R is mediated by calmedin (16) or calmodulin (CaM) (17, 18). Nevertheless, the molecular identity of calmedin has not been established, and recent results strongly suggest that high [Ca2+]i inhibition of the InsP3R is not mediated by CaM (19–21).

We previously identified a biochemical and functional interaction between the InsP3R and a family of CaM-related Ca2+-binding proteins (CaBPs) (22), a subset of the neuronal Ca2+ sensor (NCS) family of EF-hand-containing proteins that contains eight genes (CaBP1–8) with alternatively spliced forms (23, 24). CaBP1 bound with high affinity (apparent KD < 50 nM) in a Ca2+-dependent manner within the NH2-terminal 600 residues of all three mammalian InsP3R isoforms, a region that encompasses the InsP3-binding domain. Of note, in single-channel electrophysiological studies, acute application of recombinant CaBP1, CaBP2, and CaBP5 activated channel gating in the absence of InsP3. In contrast, CaM neither bound with high affinity nor activated channel gating (22). These studies therefore identified CaBPs as specific protein ligands of the InsP3R channel.

The Ca2+ dependence of the interaction with CaBP1 suggested that the InsP3R could possibly become engaged in vivo as a Ca2+-induced Ca2+ release channel in the absence of InsP3 generation and that the interaction might tune the sensitivity of the channel to InsP3 (22). Subsequent studies revealed that overexpression of CaBP1 attenuated InsP3R-dependent Ca2+ release in intact cells (25, 26). Taken together, the results appear to suggest that CaBPs can tune InsP3R activity by both activation and inhibition. However, the mechanisms that enable these proteins to both activate and inhibit the channel are not clear.

CaBPs are exclusively expressed in the brain and retina (24). However, it is unknown whether non-neuronal proteins exist that function as protein ligands of the InsP3R in peripheral tissues. We reasoned that more widely expressed, structurally similar EF-hand proteins might also interact with InsP3Rs as channel ligands. To facilitate their identification, we have first characterized the structural determinants for CaBP1 binding to the InsP3R. We found that disruption of any one of three functional EF-hands reduced binding to the InsP3R, with EF3 and EF4 being particularly important. Based on these observations, we examined other ER-localized proteins with functional EF3 and EF4 for binding to the InsP3R. We show that one of these, CIB1 (calcium- and integrin-binding protein; also called calmyrin or KIP), a widely expressed protein, bound strongly in aCa2+-dependent manner within the InsP3-binding region of the InsP3R. In single channel experiments, CIB1 was found to act as a direct activating ligand of the channel in the absence of InsP3. However, pre-exposure of the receptor to CIB1 reduced the number of channels available for subsequent activation by InsP3, and overexpression of CIB1 decreased the amplitude of agonist-induced [Ca2+]i transients in intact cells. Our data identify CIB1 as a widely expressed novel protein ligand of the InsP3R that gates the channel and inhibits subsequent InsP3-dependent stimulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—COS-7 (Cercopithecus aethiops kidney) and HeLa cells were grown in Dulbecco's modified Eagle's medium/high glucose medium containing 10% (v/v) fetal bovine serum (Invitrogen), 100 units/ml–1 penicillin, and 100 µgml–1 streptomycin. PC12 cells were grown in F-12K medium (ATCC) supplemented with 15% (v/v) horse serum (Invitrogen), 2.5% (v/v) fetal bovine serum, and 100 µgml–1 penicillin and 100 µg ml–1 streptomycin and maintained in a humidified 95, 5% air, CO2 atmosphere. Spodoptera frugiperda (Sf9) cells were maintained in suspension culture at 27 °C in serum-free Sf-900 II SFM medium (Invitrogen).

Molecular Biology and Biochemistry—Cloning of CaBP1 short (GST-s-CaBP1) and NH2-terminally truncated s-CaBP1 (GST-c-CaBP1) into pGex-6P-1 (Amersham Biosciences), and subsequent generation of GST fusion proteins has been described (22, 24). Using GST-c-CaBP1 as template, EF-hand mutants were generated with the QuikChangeTM site-directed mutagenesis kit (Stratagene). Truncation mutants were generated from full-length s-CaBP1 in pGEX-6P-1 as template. CIB1 was subcloned from pBluescript KS(–) (27) into pGex-6P-1, which was then used as template to generate EF-hand mutants (primer sequences available upon request for all reagents described). The NH2-terminal 521 residues of rat InsP3R-2 were cloned into pcDNA/V5 (Invitrogen). Other InsP3R constructs have been described (22). GST fusion proteins were expressed in Escherichia coli (BL-21; Stratagene), and pull-down assays were performed as described (22). Co-immunoprecipitation and Western blot analyses were performed according to standard protocols. To generate untagged CIB1, GST was cleaved from recombinant fusion protein by PreScission protease (Amersham Biosciences) and further purified according to the manufacturer's instructions.

Electrophysiology of InsP3R—Patch clamp experiments were performed on isolated Xenopus oocyte (5, 13) and Sf9 cell (28) nuclei as described. Sf9 cells were washed twice with phosphate-buffered saline and suspended in a nuclear isolation solution containing (in mM): 150 KCl, 250 sucrose, 1.5 beta-mercaptoethanol, 10 Tris-HCl, 0.05 phenylmethylsulfonyl fluoride, protease inhibitor mixture (Complete, Roche Applied Science), pH 7.5. Nuclei were isolated using a Dounce glass homogenizer and plated onto a 1-ml glass-bottomed dish containing standard bath solution (in mM): 140 KCl, 10 HEPES, and 0.5 BAPTA (free [Ca2+] = 50–100 nM), pH 7.1. The pipette solution contained (in mM): 140 KCl, 0.5 ATP, 10 HEPES, pH 7.1. Free [Ca2+] was adjusted by the addition of appropriate Ca2+ chelators (5, 13) and routinely measured by indicator dye fluorescence. Experiments were performed at room temperature. Data were acquired using an Axopatch-1D amplifier (Axon Instruments) as described (5). Segments of current traces exhibiting one InsP3R channel were used for open probability (Po) determinations (29), and single channel analysis was performed using the QuB software (30). Particular consideration was given to the accurate determination of the number of active channels in nuclear membrane patches (NA) from the experimental current records, using the set of criteria described in Ref. 28.

Calcium Imaging of Transfected Cells—CIB1, NCS-1, and s-CaBP1 cDNAs were cloned into pIRES2-EGFP (Clontech) and electroporated (Nucleofector device, Amaxa) according to the manufacturer's instructions. Transfected cells were seeded onto glass coverslips, cultured for 48 h, transferred to a perfusion chamber on the stage of a microscope (Nikon TE2000), and incubated with fura-2 AM (Molecular Probes; 2 µM) for 60 min at room temperature in normal medium. Cells were continuously perfused with Hanks' balanced salt solution (Sigma) containing 1.8 mM CaCl2 and 0.8 mM MgCl2, pH 7.4. Transfected cells were identified by green fluorescent protein fluorescence; fura-2 was alternately excited at 340 and 380 nm, and emitted fluorescence (510 nm) was collected and recorded using a CCD-based imaging system running Ultraview software (PerkinElmer Life Sciences). Dye calibration was achieved by applying experimentally determined constants to the equation: [Ca2+] = Kd · beta · (R – Rmin)/(Rmax – R).

Analysis and Statistics—Data were summarized as mean ± S.E.; statistical significance of differences between means, assessed using unpaired t-tests, were accepted at the 95% level (p < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structural Determinants of CaBP1 Binding to InsP3R—Both long and short NH2-terminal splice variants of CaBP1 (l-CaBP1 and s-CaBP1) bind with high affinity within the InsP3-binding NH2-terminal 600 residues of the InsP3R. The interaction was strongly potentiated by Ca2+ (Refs. 22 and 25, although see Ref. 26), due in part to Ca2+ binding to CaBP1, because a CaBP1 mutant protein with all three functional EF-hands disabled failed to interact. A construct (c-CaBP1) encompassing the COOH-terminal region containing all four EF-hands that is shared among the splice variants bound nearly as well as s-CaBP1 (Fig. 1A). Deletion of EF-hands 1 and 2 (EF1, EF2), creating a truncated protein identified as calbrain (31), decreased binding efficiency by ~50% (Fig. 1A). The substantial binding of this construct suggested that functional EF-hands 3 and 4 are important for InsP3R binding. Disabling EF1, by replacements with alanines at positions one and three, reduced InsP3R binding by ~25% (Fig. 1B), whereas mutating either EF4 or EF3 reduced binding by ~46 and ~75%, respectively (Fig. 1B). Although some binding was observed to c-CaBP1 with only EF3 functional, mutation of EF3 and either EF1 or EF4 or all three EF-hands together eliminated binding (Fig. 1B). These data suggest that there is no strict requirement for specific EF-hands but that at least two are necessary, with Ca2+-binding EF3 and EF4 of particular importance.


Figure 1
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  		FIGURE 1.
Binding of CaBP1 deletion- and EF-hand-mutants (mEF) to InsP3RNH2 terminus. CaBP1 deletion (A) and c-CaBP1 EF-hand mutant (B) constructs represented schematically with functional (solid rectangle), non-functional (open rectangle), and mutated (gray rectangle) Ca2+-binding EF-hands are shown. Lysates of Sf9 cells expressing V5-tagged NH2-terminal 600 residues of InsP3R-1 were incubated with GST-CaBP1 fusion proteins. The total amount of fusion protein in the reaction was assessed by anti-GST Western blot (A, left lane labeled input) or Coomassie Blue staining (B, left lane labeled input); bound InsP3R was detected with anti-V5 antibody (right lane labeled pull-down). The amount of InsP3R bound to GST-CaBP1 constructs was determined by quantitative densitometry and normalized to pull-down by GST-s-CaBP1 (A)(n = 3) or c-CaBP1 (B)(n = 4). Data plotted are mean (±S.E.).

 
Identification and Characterization of CIB1 as an InsP3R Ligand-binding Domain-interacting Protein—We hypothesized that other ER-localized proteins with functional EF-hands 3 and 4 might also interact with the InsP3R. We examined four candidates: sorcin, a penta-EF-hand protein that interacts with the ryanodine receptor Ca2+ release channel (32, 33); CIB1 (calmyrin, KIP), a ubiquitously expressed protein with homology to calcineurin B (57%) and calmodulin (54%) (34) that has only EF3 and EF4 of its four EF-hands functional for Ca2+ binding; and calsenilin (KChIP3, DREAM) and NCS-1 (frequenin), two members of the NCS family that have EF1 non-functional for Ca2+ binding (35) (Fig. 2A). CaM, which contains four functional EF-hands, interacts only weakly with the InsP3R ligand-binding domain, with an apparent affinity over an order of magnitude weaker than that of CaBP1 (22). Similarly, neither sorcin, calsenilin, nor NCS-1 interacted with the channel (Fig. 2B). In contrast, CIB1 was very efficient in pulling down the InsP3R-1 NH2-terminal domain from Sf9 cell lysates (Fig. 2B), although its affinity appears to be somewhat weaker than that of CaBP1. These results demonstrate specificity in the interactions of the InsP3R with EF-hand-containing proteins and establish CIB1 as a protein interactor of the ligand-binding region of the InsP3R.

GST-CIB1 effectively pulled down endogenously expressed InsP3R-3 from lysates of COS-7 cells and whole rat lungs (Fig. 3A), as well as an expressed rat InsP3R-2 InsP3-binding region from COS-7 cell lysates (Fig. 3A). Thus, CIB1 interacts with all three InsP3R isoforms. Immunoprecipitation of endogenous InsP3R-3 co-precipitated endogenous CIB1 from COS-7 and HeLa cells (Fig. 3B), suggesting that the endogenous proteins can interact in vivo.

Ca2+ binding to EF-hand-containing proteins, including CaBP1 (36) and CIB1 (37), induces conformational changes that modulate target protein interactions (35, 38). The [Ca2+] dependence of CaBP1 binding to InsP3R was examined previously by quantifying binding of GST-CaBP1 to endogenous COS-7 cell InsP3R-3 in lysates with [Ca2+] fixed over a wide range (22). We assessed the [Ca2+] dependence of the CIB1-InsP3R interaction using the same methods. Increasing [Ca2+] from 10 nM to 100 µM strongly enhanced binding by over 10-fold with apparent half-maximal activity at ~1–5 µM (Fig. 4A).


Figure 2
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  		FIGURE 2.
Interaction of InsP3R-1 with EF-hand-containing proteins. A, domain structures of EF-hand-containing proteins showing functional Ca2+-binding (filled rectangles) and non-functional (open rectangles) EF-hands. B, lysates of Sf9 cells expressing V5-tagged NH2-terminal 600 residues of InsP3R-1 (1–600-InsP3R-1) were incubated with various EF-hand-containing GST fusion proteins. Only s-CaBP1 and CIB1 pulled down the InsP3R (upper gel). Coomassie Blue staining of GST fusion proteins (lower gel) was used to equalize the amount of GST fusion protein used in each in vitro binding reaction. Results are representative of three experiments.

 


Figure 3
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  		FIGURE 3.
CIB1 interaction with type-2 and type-3 InsP3R. A, endogenous InsP3R-3 in lysates from COS-7 cells (left) and rat lung (right) and V5-tagged NH2-terminal 521 residues of type-2 InsP3R (1–521-InsP3R-2) expressed in COS-7 cells (lower) were all effectively pulled down by GST-CIB1. Results are representative of four experiments. B, immunoprecipitates (IP) from COS-7 (upper gel) and HeLa (lower gel) cells probed with mouse anti-InsP3R-3 antibody, revealing specific co-immunoprecipitation of endogenous InsP3R-3 with CIB1. Results are representative of two experiments.

 


Figure 4
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  		FIGURE 4.
[Ca2+] dependence of InsP3 R-CIB1 interaction. A, GST-CIB1 pull-down of endogenous InsP3R-3 from COS-7 cell lysates in various free [Ca2+] adjusted to the desired level by varying Ca2+/EGTA, calculated using maxchelator (C. Patton, Stanford University, CA). Results are representative of two experiments. B, endogenous InsP3R-3 is less effectively pulled down by GST-CIB1 mutants with EF3 or EF4 non-functional (upper gel). Coomassie Blue staining confirmed that equivalent amounts of protein were present (lower gel). Results are representative of five experiments. wt, wild type.

 
The similar [Ca2+] dependences of the interactions of CIB1 and CaBP1 with the InsP3R and the dependence on functional EF-hands in CaBP1 for the interaction suggested that the Ca2+-binding EF-hands in CIB1 are important for the interaction (Fig. 1). Disruption of either EF-hand profoundly reduced the interaction between CIB1 and InsP3R-3 (Fig. 4B), confirming their functional importance. In summary, CIB1 interacts with the ligand-binding region of all three isoforms of the InsP3Rin a Ca2+-dependent manner that depends on Ca2+ binding to both functional EF-hands, features all highly reminiscent of the interaction of CaBP1 with the InsP3R.

CIB1 Is a Protein Ligand of the InsP3R Channel—We showed previously that in vitro interaction of CaBP1 with the InsP3R activated single channel gating in the absence of added InsP3 (22). The analogous biochemical interactions of CaBP1 and CIB1 with the InsP3R suggested that CIB1 might also be functionally active. Single-channel activity of endogenous Xenopus InsP3R-1 was recorded by patch clamp electrophysiology of outer membranes of nuclei isolated from Xenopus oocytes (5, 13). Robust channel activity (open probability, Po {approx} 0.75) was observed with pipette solutions containing 10 µM InsP3 and 20 µM Ca2+ to optimally activate gating (Fig. 5A) (13). Active channels were detected in 75% of patches, with mean number of active channels per patch (NA) {approx} 2.3. Inclusion of 1 µM recombinant CIB1 (r-CIB1) in the pipette solution in lieu of InsP3 activated InsP3R channels with Po {approx} 0.5 and NA {approx} 0.5. A higher concentration (10 µM) did not elicit further increases (Fig. 5B). Channel activation required CIB1 binding since neither mutant CIB1 with either EF-hand disrupted activated gating (Fig. 5A). These data establish CIB1 as a protein ligand of the channel, although CIB1 seems less efficacious than InsP3 since it activated fewer channels (NA) with lower activity (Po) when compared with InsP3 (Fig. 5B). The total InsP3R-mediated ion flux across the ER membrane is the product of single-channel conductance, Po and NA. Because single-channel conductance remained constant under the present experimental conditions, NAPo provides a measure of the total ion flux associated with InsP3R activation. NAPo activated by CIB1 was less than 20% of that achieved by saturating [InsP3] (Fig. 5B), suggesting that CIB1 functions as a weak or partial agonist of the channel.


Figure 5
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  		FIGURE 5.
Effect of recombinant CIB1 on single-channel InsP3R activity recorded in nuclear envelope patches from Xenopus oocytes. A, typical InsP3R single-channel current recordings (applied potential, 20 mV) in the presence of saturating (10 µM) InsP3; 1 µM r-CIB1 (purity shown in Supplementary Fig. 2); or mutant EF-hand 3 or 4 CIB1 (mEF3 or mEF4; 1 µM). Pipette [Ca2+] was 20 µM; the arrows indicate current levels. B, with 10 µM InsP3, Po was 0.74 ± 0.03 (mean ± S.E.; n = 14); in 1 or 10 µM r-CIB1, it was 0.53 ± 0.08 (n = 7) and 0.41 ± 0.09 (n = 6), respectively (n = number of patches used in Po determination). Mean number of activated channels (NA) in 10 µM InsP3 was 2.3 ± 0.2 (n = 63); in 1 or 10 µM r-CIB1, it was 0.54 ± 0.1 (n = 50) and 0.67 ± 0.2 (n = 24), respectively. No channel activity was observed in 15/15 or 16/17 patches in the presence of mEF3 r-CIB1 or mEF4 r-CIB1, respectively. Total ER ion flux (NAPo) was lower in the presence of r-CIB1 when compared with InsP3. Asterisks, p < 0.01 (**) and p < 0.05 (*), unpaired t test.

 
CIB1 Inhibits InsP3R-dependent Ca2+ Signaling in Intact Cells—The above results demonstrated that, at the molecular level in vitro, Ca2+-dependent binding of CIB1 to the InsP3R results in channel activation, reminiscent of the effects of CaBP1 (22). Activation of InsP3R by CaBP1 was subsequently questioned in two studies reporting that CaBP1 overexpression attenuated, rather than stimulated, InsP3R-dependent [Ca2+]i signaling in intact cells (25, 26). Nevertheless, we show that another EF-hand-containing protein similarly binds to and activates InsP3R channel gating. The reasons for the seemingly conflicting results obtained in in vitro electrophysiological experiments and intact cells are not obvious. We therefore examined the effects of CaBP1 and CIB1 expression on [Ca2+]i signaling to determine whether inhibitory effects observed in the other studies could be reproduced and whether CaBP1 and CIB1 have similar effects.

We first examined the PC12 cell neuroendocrine cell line, in which it was reported that CaBP1 expression inhibited InsP3R-dependent [Ca2+]i signaling (25). Transient expression of either s-CaBP1 or CIB1 reduced the peak amplitude of the [Ca2+]i transients elicited by 10 and 30 µM ATP by ~50% when compared with cells expressing the empty vector (Fig. 6, A–D). In contrast, there were no differences in cells expressing NCS-1 (Fig. 6, B and D), which does not bind to the InsP3R (Fig. 2B) (25) but has similar Ca2+ binding properties to CaBP1 (25), indicating that changes in cellular Ca2+-buffering capacity cannot account for the observed inhibition. Caffeine-induced [Ca2+]i transients mediated by ryanodine receptor activation were unaffected by expression of either CaBP1 or CIB1 (Fig. 6E), suggesting that the diminished ATP responses were not related to reduced stores of intracellular Ca2+ and were specific for the InsP3R pathway. The store content was independently evaluated by measuring peak [Ca2+]i responses in cells exposed to the Ca2+ ionophore ionomycin in Ca2+-free medium. Neither s-CaBP1 nor CIB1 expression reduced the total availability of stored Ca2+, as assessed by this protocol (Fig. 6F). The total InsP3R protein content of the cells was unaffected by overexpression of s-CaBP1, CIB1, or NCS-1, indicating that the observed inhibition of [Ca2+]i signaling could also not be accounted for by reduced InsP3R expression levels (Fig. 6G). Similar results were obtained when s-CaBP1 and CIB1 were transiently expressed in HeLa cells, the other cell type employed in the Haynes et al. study (25) (Supplementary Fig. 1).


Figure 6
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  		FIGURE 6.
Effects of CIB1 or s-CaBP1 expression on agonist-evoked [Ca2+]i transients in PC12 cells. A, representative single cell [Ca2+]i transients evoked by 10 µM ATP in PC12 cells expressing NCS-1, s-CaBP1, CIB1, or vector alone. B, summary (mean ± S.E.) of peak transients in cells expressing indicated proteins. Peak amplitude in control and s-CaBP1- and CIB1-expressing cells: 517 ± 48 nM, 318 ± 40, 274 ± 35 nM, respectively. C, typical transients evoked by 30 µM ATP in same group of cells. D, peak amplitude in vector-expressing control cells was 1500 ± 159 nM; it was reduced to 727 ± 102 and 868 ± 90 nM in cells expressing s-CaBP1 and CIB1, respectively. Summary data from at least 30 cells from multiple coverslips in two independent trials. Asterisks, p < 0.01 (*), unpaired t test. E, peak amplitude (mean ± S.E.) in response to 10 mM caffeine in cells expressing s-CaBP1 (n = 24), CIB1 (n = 15), or vector (n = 16) alone. F, peak amplitude (mean ± S.E.) in response to 5 µM ionomycin. G, InsP3R-3 expression by Western blot of lysates from cells expressing vector, NCS-1, s-CaBP1, or CIB1.

 
The inhibitory effects we have observed are reminiscent of the reported effects of CaBP1 expression on [Ca2+]i signals in PC12 and HeLa cells (25) and in COS-7 cells (26). Furthermore, we have now extended those observations by demonstrating that CIB1 impinges on InsP3R-dependent signaling in a similar manner. We conclude that although CaBP1 and CIB1 behave as activating protein ligands of the InsP3Rin in vitro single channel studies, they behave as antagonists of the InsP3-dependent [Ca2+]i signaling pathway when overexpressed in intact cells.

CIB1 Decreases the Number of Channels Available for InsP3 Activation—The effects of CaBP1 and CIB1 are paradoxical. On the one hand, their overexpression in intact cells inhibits InsP3R-mediated Ca2+ release, whereas both proteins activate Ca2+ release in vitro at the single channel level. How can CaBP1 and CIB1 be inhibitory in vivo yet activating in vitro?

One fundamental difference in the two experimental approaches is the kinetic resolution of the assays with respect to the initial interaction between the Ca2+-binding proteins and the InsP3R. The electrophysiology experiments measure the earliest response of the channel to interaction with the protein in the absence of InsP3, whereas in vivo imaging of transfected cells examines the responses of the InsP3R to InsP3 at unknown and variable amounts of time after, or during, the interaction of CaBP1/CIB1 with the InsP3R. Following initial activation of the channel by InsP3, a process of ligand-dependent inactivation has been proposed to explain Ca2+ release termination in the presence of constant ligand concentrations (6, 39–41). InsP3-dependent single-channel inactivation has been reported for Xenopus InsP3R-1 (29) and Sf9 cell InsP3R (28) channels. Furthermore, we observed abrupt InsP3R channel activity termination following activation by either CaBP1 or CIB1 (not shown). We therefore considered the possibility that binding of CaBP or CIB1 initially activates InsP3R gating (as observed in single channel experiments), but the channel then undergoes ligand-dependent inactivation, rendering it refractory to subsequent InsP3 stimulation (as observed in the intact cell studies). A mechanism of protein ligand-induced InsP3R channel inactivation could therefore reconcile the seemingly disparate results obtained in electrophysiological and intact cell experiments.

To test this hypothesis, we attempted to recreate the long term interaction of the protein ligands with the InsP3R in the intact cell experiments in vitro by exposing the InsP3R to CIB1 prior to InsP3 application. For these experiments, InsP3R channel activity was studied by patch clamp electrophysiology of nuclei isolated from insect Sf9 cells, believed to express only one InsP3R isoform. The Sf9 system is ideal for studies of InsP3R gating kinetics, including inactivation, because channels are consistently detected and their mean activity duration is much longer than in Xenopus oocyte nuclei (28). In control experiments, multiple channels (NA {approx} 4) with high Po (~0.6) were observed with pipettes containing 100 nM InsP3 and 1 µM Ca2+ (Fig. 7, A and B), whereas less robust activity was recorded with r-CIB1 (1 µM) included in the pipette in lieu of InsP3 (Po {approx} 0.35, NA {approx} 0.9; Fig. 7, A and B), consistent with the less robust responses to CIB1 of the Xenopus InsP3R channel (Fig. 5). To mimic in vitro the effect of CIB1 expression in intact cells, isolated nuclei were preincubated with 5 µM CIB1 for 20–30 min in a bath solution containing 50–100 nM Ca2+, and subsequently patched with pipettes containing 100 nM InsP3 and 1 µM Ca2+ (Fig. 7A). Channel Po was not altered when compared with controls, whereas NA was decreased by nearly half (Fig. 7B). The product NA Po was correspondingly reduced (Fig. 7B), indicating that the ER ion flux was reduced ~50% by preincubation with CIB1. Simultaneous exposure of the channel to InsP3 and CIB1 did not reduce NA or Po (not shown), indicating that the pre-exposure was required.


Figure 7
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  		FIGURE 7.
Effect of recombinant CIB1 on single-channel InsP3R activity recorded in nuclear envelope patches from Sf9 cells. A, typical InsP3R single-channel current recordings (applied potential 20 mV) in the presence of 100 nM InsP3, 1 µM r-CIB1, or 100 nM InsP3 after preincubation with 5 µM r-CIB1. Pipette [Ca2+] was 1 µM; the arrows indicate current levels. B, with pipettes containing 100 nM InsP3, Po was 0.60 ± 0.06 (mean ± S.E.; n = 10 patches used in Po determination); with 1 µM r-CIB1, it was 0.35 ± 0.14 (n = 5). Mean number of activated channels (NA) in 100 nM InsP3 was 3.86 ± 0.4 (n = 14); in 1 µM r-CIB1, it was 0.88 ± 0.3 (n = 8). In nuclei preincubated with r-CIB1, Po and NA in response to 100 nM InsP3 were 0.48 ± 0.07 (n = 13) and 2.20 ± 0.4 (n = 15), respectively. The product NAPo represents an index of total ER ion flux. Asterisks, p < 0.01 (*), unpaired t test.

 
These data suggest that pre-exposure to CIB1 renders some channels unavailable for activation by InsP3, consistent with the hypothesis that prolonged exposure in vivo induces channel inactivation, causing the subsequent responses to InsP3-generating agonists to be muted. The extent of channel inhibition we observed in these in vitro experiments is similar to the magnitude of the inhibition of [Ca2+]i signaling observed in this work (Fig. 6) and previously in response to CaBP1 (25). The fact that it was still possible to activate some channels with InsP3 after CIB1 pre-exposure suggests that not all channels had been driven into an inactivated state, consistent with the lower efficacy of CIB1 to recruit channels into activity (Figs. 5B and 7B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
InsP3R-mediated Ca2+ signals are shaped in part by messenger generation, diffusion, degradation and removal, and kinetics of interaction with the channel, processes that are expected to vary greatly among different modulators of the release channel. Thus, the discovery of CaBPs as direct protein ligands of the InsP3R extended existing concepts of the dimensions and versatility of the InsP3R-dependent signaling system. However, the neuronally restricted expression of CaBP proteins raised the question of whether this new mode of regulation and [Ca2+]i signaling extended more widely to non-neuronal cell types as well. We used structure-function analysis of the sequence determinants in CaBP1 that mediate its biochemical interaction with the InsP3R to guide a directed search for other EF-hand-containing protein interactors of the channel. We identified CIB1 as a widely expressed CaM-related protein that shares many of the properties of CaBP1 with respect to its interactions with the InsP3R. Thus, CIB1 binds to the first 600-residue ligand-binding region of all isoforms of the InsP3R in a Ca2+-sensitive fashion dependent on functional Ca2+-binding EF-hands. Furthermore, purified CIB1 activates gating of the InsP3R in the absence of InsP3. These results extend the concept of protein ligands of the InsP3R to peripheral tissues and suggest that regulation of the InsP3R-mediated [Ca2+]i signaling may be under complex regulation by protein ligands in many cell types throughout the body.

Previous studies revealed the interaction of CaBP1 with the InsP3R to be strongly potentiated by Ca2+, with apparent Ca2+ affinity between 0.1 and 1 µM (Refs. 22 and 25, although see Ref. 26), dependent on functional EF-hands in CaBP1 (22). Using site-directed mutagenesis and protein truncations, we found that functional Ca2+-binding EF-hands 3 and 4 are particularly important in mediating the Ca2+ dependence. Recent structural analysis of CaBP1 confirmed that EF2 does not bind divalent metal ions and showed that EF1 has a low Ca2+ affinity (apparent KD of 300 µM) with little selectivity between Ca2+ and Mg2+, whereas Ca2+ binds cooperatively to EF3 and EF4 with an apparent affinity of 2.5 µM (36). These structural results are in good accord with binding data in the present and previous studies (22). Ca2+ binding to CaBP1 induces a conformational change specifically in the COOH-terminal region containing EF3 and EF4 (36). Together, these results suggest that the Ca2+ dependence of the interaction of CaBP1 with the InsP3R is mediated by cooperative Ca2+ binding to EF3 and EF4 that induces localized conformational changes in the COOH-region of CaBP1 that facilitates its binding to the channel.

Based on these insights, we analyzed other EF-hand-containing proteins to discover whether additional ones could also interact with the InsP3R. Although many proteins contain COOH-terminal pairs of EF-hands, we restricted our analysis to those previously shown to interact with ER- or Golgi-localized proteins. Of the relatively few proteins examined, only CIB1 interacted with the InsP3R. The lack of interaction of the channel with NCS-1 confirms previous results (25), and the specific interaction with CIB1 demonstrates specificity in the interactions of EF-hand-containing proteins with the ligand-binding region of the InsP3R.

Structural analyses have placed CIB1 and CaBP1 in distinct subfamilies of NCS proteins (34). CIB1 is myristoylated at its NH2 terminus, which localizes it to cell membranes (27, 42), and both EF-hand-containing lobes are positioned in close contact to each other and bind to the same side of the substrate (34). Unlike CaBPs, which occur almost exclusively in the nervous system, CIB1 is ubiquitously expressed (42), where it has been shown to interact with a host of diverse target proteins in peripheral tissues (see Ref. 34 for references). We found that CIB1 binds to all three InsP3R isoforms within the same ligand-binding domain as CaBP1 and can be co-immunoprecipitated with InsP3R from cell lysates. Like CaBP1, CIB1 binding to InsP3R has a similar dependence on functional EF-hands 3 and 4. CIB1 EF-hands 3 and 4 bind Ca2+ with Kd of 0.5–2 µM Ca2+ (36, 43), which correlates well with the Ca2+ dependence of its interaction with the InsP3R observed in this work.

The crystal structure of human CIB1 (34, 44) shows it to be a compact {alpha}-helical protein with two Ca2+ ions bound in a canonical fashion by the last two EF-hands. It is structurally similar to calcineurin B, calcineurin B homologous protein 1 (CHP1), and KChIP1 (34), which are in turn believed to be representative of the entire family of NCS proteins, with folds distinctly different from those of CaM. Like NCS proteins, CIB1 contains a hydrophobic pocket on one surface of the protein opposite from that of the Ca2+-binding sites. This pocket interacts with hydrophobic residues in amphipathic {alpha}-helices of interacting partners (45, 46). It is likely that CaBP proteins adopt similar folds and interact with target ligands in a similar fashion. These considerations suggest that amphipathic helices in the NH2 terminus of the InsP3R likely mediate its binding to CaBP1 and CIB1. Within the 600 NH2-terminal residues of the InsP3R, an NH2-terminal beta-sheet-rich beta-trefoil domain contains an unusual helix-loop-helix insertion (H2 and H3) (47). Based on in vitro binding to synthesized small peptides, it was proposed that CaBP1 interacted with residues Pro-49–Asn-81 (26) within this region. In the crystal structure, the distal end of this region contains H2, possibly implicating it in the interaction with CaBP1/CIB1. However, the interaction with the peptide was Ca2+-independent, whereas the interaction of both CaBP1 (22, 25) and CIB1 (this study) are strongly Ca2+-sensitive. Furthermore, the peptide interacted with comparable affinity as CaM, whereas the interaction of CaBP1 with the InsP3R has over an order of magnitude higher affinity than that of CaM (22). Thus, the relevance of this region for the Ca2+-dependent, high affinity interaction of CaBP1/CIB1 with the InsP3R is unclear. The region encompassing residues 225–604, which includes the core InsP3-binding domain, contains a distal armadillo-repeat domain (48). It is possible that CaBP1/CIB1 interact with the channel by binding to an {alpha}-helix in this region.

The functional consequences of the CIB1-InsP3R interaction were determined by recording single InsP3R channels in nuclei isolated from either Xenopus oocytes or Sf9 cells. With optimal [Ca2+] and in the absence of InsP3, CIB1 activated channel gating from both species, establishing it as a novel protein agonist. When compared with optimal [InsP3], however, CIB1 activated fewer channels to a lesser extent in both the oocyte and the Sf9 systems. Thus, CIB1 appears to behave as a partial agonist of the channel.

Despite the ability of CaBP1 to act as an activating ligand of the InsP3R channel when applied acutely in patch clamp studies (22), it was subsequently shown that its overexpression inhibited InsP3R-dependent Ca2+ release in cells (25, 26). Nevertheless, the discovery that another protein, CIB1, behaves in patch clamp studies similarly to CaBP1 reinforces the validity of those previous studies. The apparent discrepancy between results from electrophysiological studies and cell expression studies prompted us to explore the possible reasons. We found that overexpression of either s-CaBP1 or CIB1 attenuated agonist-mediated, InsP3-dependent Ca2+ release, which could not be accounted for by reduced store content, diminished InsP3R expression levels, or increased cytoplasmic Ca2+ buffering. Our results are therefore in accord with the previous reports (25, 26) of the effects of CaBP1 on [Ca2+]i signaling.

Several possible mechanisms might reconcile that, on the one hand, acute exposure of the channel to purified recombinant CaBP/CIB1 proteins activates channel gating, whereas their overexpression mutes InsP3R-dependent [Ca2+]i signals in cells. First, in intact cells, CaBP1 or CIB1 might bind to the InsP3R even under conditions of resting [Ca2+]i by avidity if the proteins are in close proximity or because of the finite ability of the proteins to bind even in low [Ca2+]i. Notably, both effects will be magnified in overexpression studies. Despite being liganded by these proteins, the channel may not be activated if the Ca2+ requirement for channel gating is not satisfied, whereas the protein-bound InsP3R might be less sensitive to InsP3. Consequently, agonist-induced [Ca2+]i signals would be inhibited, as observed (present results and Refs. 25 and 26). Second, because InsP3 binding is believed to drive the channel into an inactive state from which it recovers only after ligand dissociation (6, 28, 39–41), we considered the possibility that CaBP/CIB1 binding to the channel initially activates it but then subsequently drives it into an inactivated state that cannot be activated by InsP3. We tested this model explicitly be pre-exposing the InsP3R channels to CIB1 and then subsequently assaying the ability of the channels to become activated by InsP3. CIB1 pre-exposure reduced the number of channels that could be activated by InsP3, although channels that were activated had normal Po. These results are consistent with CIB1-induced inactivation of a subset of the total channel population, with the remaining channels that had not been activated by the pre-exposure able to respond normally to InsP3. In intact cells, the inactivated channels would remain inactivated as long as they were liganded by CIB1. Two factors might retain much of the InsP3R channel population in an inactivated state in vivo. First, high [CaBP1] as a consequence of overexpression would ensure a high probability of binding to the InsP3R. Second, slow unbinding of the NCS proteins from the channel may keep many channels inactivated even in the absence of overexpression. The data suggest that exposure to CIB1 can effectively remove functional channels from the total InsP3R population by a process of ligand-induced channel inactivation. In vivo, this would result in fewer channels available to respond to agonist-induced increases in [InsP3], with consequent muted [Ca2+]i signals, as observed in this study and previously (25, 26)).

Our results indicate that CaBP and CIB1 can function in dual roles, as both activators and inhibitors of the InsP3R. Cellular mechanisms might exist that regulate protein ligand dissociation from the channel and enable it to escape from inactivation. Phosphorylation of CaM decreases its affinity for target substrates (49), and mutation of the conserved phosphorylation site in CaBP1 modulated [Ca2+]i signaling inhibition (26). Although this site is not conserved in CIB1, it is possible that covalent modifications or protein interactions could regulate the interactions of CaBP1 and CIB1 with the InsP3R. Escape from inactivation may then enable the channel to become activated by protein ligand rebinding.

In conclusion, we have identified a novel interaction between the widely expressed Ca2+-binding protein CIB1 and the InsP3R. Taken together, our data support a model in which CaBP1 or CIB1 serve as both positive and negative regulators of InsP3R function and extend the concept of protein ligand regulation of InsP3R function from neuronal tissues to peripheral ones as well. Future studies will be required to determine roles of these interactions in modulating the InsP3R-dependent [Ca2+]i signaling system in different cell types under a variety of physiological conditions.


   FOOTNOTES
 
* This work was supported by Grant R01-GM056328 from the National Institutes of Health (to J. K. F.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures. Back

1 To whom correspondence should be addressed: Dept. of Physiology, University of Pennsylvania, B39 Anatomy-Chemistry Bldg., 414 Guardian Dr., Philadelphia, PA 19104-6085; Tel.: 215-898-1354; Fax: 215-573-6808; E-mail: foskett{at}mail.med.upenn.edu.

2 The abbreviations used are: ER, endoplasmic reticulum; InsP3, 1,4,5-trisphopshate; InsP3R, inositol 1,4,5-trisphopshate receptor; CaM, calmodulin; CIB1, calcium- and integrin-binding protein; r-CIB1, recombinant CIB1; NCS, neuronal Ca2+ sensor; GST, glutathione S-transferase; CaBP, Ca2+-binding protein. Back


   ACKNOWLEDGMENTS
 
For providing reagents, we thank Dr. A. Jeromin (NCS-1 cDNA), Dr. J. Buxbaum (calsenilin cDNA), Dr. T.-W. Kim (sorcin cDNA), Dr. J. F. DuFour (InsP3R-2 cDNA), and Dr. S. Joseph (InsP3R antibody). We thank Drs. D.-O. D., Mak and K.-H. Cheung for comments on the manuscript.



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 DISCUSSION
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