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. 2002 May 28;99(11):7711-6.
doi: 10.1073/pnas.102006299.

Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca(2+) release channels

Jun Yang  1 , Sean McBride, Don-On Daniel Mak, Noga Vardi, Krzysztof Palczewski, Françoise Haeseleer, J Kevin Foskett
Affiliations

Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca(2+) release channels

Jun Yang et al. Proc Natl Acad Sci U S A. .

Abstract

The inositol trisphosphate (InsP(3)) receptor (InsP(3)R) is a ubiquitously expressed intracellular Ca(2+) channel that mediates complex cytoplasmic Ca(2+) signals, regulating diverse cellular processes, including synaptic plasticity. Activation of the InsP(3)R channel is normally thought to require binding of InsP(3) derived from receptor-mediated activation of phosphatidylinositol lipid hydrolysis. Here we identify a family of neuronal Ca(2+)-binding proteins as high-affinity protein agonists of the InsP(3)R, which bind to the channel and activate gating in the absence of InsP(3). CaBP/caldendrin, a subfamily of the EF-hand-containing neuronal calcium sensor family of calmodulin-related proteins, bind specifically to the InsP(3)-binding region of all three InsP(3)R channel isoforms with high affinity (K(a) approximately 25 nM) in a Ca(2+)-dependent manner (K(a) approximately 1 microM). Binding activates single-channel gating as efficaciously as InsP(3), dependent on functional EF-hands in CaBP. In contrast, calmodulin neither bound with high affinity nor activated channel gating. CaBP1 and the type 1 InsP(3)R associate in rat whole brain and cerebellum lysates, and colocalize extensively in subcellular regions in cerebellar Purkinje neurons. Thus, InsP(3)R-mediated Ca(2+) signaling in cells is possible even in the absence of InsP(3) generation, a process that may be particularly important in responding to and shaping changes in intracellular Ca(2+) concentration by InsP(3)-independent pathways and for localizing InsP(3)-mediated Ca(2+) signals to individual synapses.

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Figures

Figure 1
Figure 1
Interaction of the InsP3R with CaBP1. (A) Domain structures of CaBPs and calmodulin. (B) Coimmunoprecipitation of CaBP1 and InsP3R-3 from control COS-7 cells (lanes 2 and 4) and COS-7 cells transiently transfected with s-CaBP1-GFP (lanes 1 and 3). Immunoprecipitates were probed with an InsP3R type 3-specific antibody (Transduction Laboratories, Lexington, KY), (Upper) or anti-CaBP1 antibody (Lower). COS-7 cells do not express endogenous CaBP1 (lane 4). The InsP3R-3 antibody does not interact with CaBP1 or GFP (not shown). (C) In vitro binding of InsP3R-3 to CaBP1 requires the NH2-terminal 600 residues of the InsP3R. Lysates from Xenopus oocytes expressing full-length r-InsP3R-3 (lane 3, lysate from 50 oocytes) or type 3 InsP3R lacking the first 600 residues (Δ1–600-InsP3R-3) (lane 2, lysate from 50 oocytes) were incubated with GST-CaBP1, and bound InsP3R was detected with a COOH-terminal InsP3R-3 antibody (49). Expression of Δ1–600-InsP3R-3 was verified by immunoprecipitation and Western blotting (lane 1, lysate from 14 oocytes). (D) All three mammalian InsP3R isoforms interact with CaBP1 in vitro. COS-7 cell lysates were incubated with GST-c-CaBP1, and bound InsP3R was detected with isoform-specific antibodies. Type 1 was pulled down with GST only (in 5 mg of lysate, lane 1) or with GST-c-CaBP1 (from 5 mg of lysate, lane 2), type 2 in GST-c-CaBP1 pull-down from 1.25 mg of lysate (lane 3), and type 3 present in 50 μg of lysate (lane 4), in pull-down with GST only (from 1.25 mg of lysate) and in pull-down with GST-c-CaBP1 (from 1.25 of mg lysate). Equivalent GST-fusion protein concentrations were present in in vitro binding reactions as shown in Right a Western blot with anti-GST antibody (Amersham Pharmacia). Intensities are within the linear range. Inspection of intensities and normalization of lysates used indicate stoichiometric interaction of InsP3R and CaBP1. (E) Homotetrameric rat types 1 and 3 InsP3R isoforms interact with CaBP1. Lysates from control COS-7 cells (−) or COS-7 cells transfected with types 3 (3, Left) or 1 (1, Right) InsP3R was incubated with GST-CaBP1, and bound InsP3R was detected with isoform-specific antibodies. Type 3 (Left): 5 μg or 250 μg of cell lysate used in first and second pairs of lanes, respectively. Type 1 (Right): 25 μg or 250 μg of cell lysate used in third and fourth pairs of lanes, respectively. Because of high level overexpression of the InsP3R, pull-down intensity is not proportional to the amount of InsP3R input in this experiment.
Figure 2
Figure 2
Ca2+ dependence of CaBP1-InsP3R interaction. (A) Elevation of [Ca2+]i enhances the interaction of the InsP3R with CaBP1. Coimmunoprecipitation, using type 3 InsP3R antibody, of CaBP1 with InsP3R-3 from lysates of CaBP1-GFP-transfected COS-7 cells (Left) exposed (+) or not (−) for 2 min to the Ca2+ ionophore ionomycin (2 μM). Immunoprecipitates (Left) or cell lysates (Right; 5 μg each) were probed for InsP3R-3 (Upper) or CaBP1 (Lower). Ionomycin enhanced the amount of CaBP1 detected in immunoprecipitates, which contained equal amounts of InsP3R (lanes 1 and 2, Top) and s-CaBP1-GFP (lanes 3 and 4, Bottom). (B) In vitro binding of InsP3R-3 to CaBP1 is specifically enhanced by Ca2+. COS-7 cell lysates, with free [Mg2+] and [Ca2+] fixed to 500 μM Mg2+/0 Ca2+ (left lane), 0 Mg2+/0 Ca2+ (center lane) or 0 Mg2+/500 μM Ca2+ (right lane) were incubated with GST-c-CaBP1, and bound InsP3R was detected with type-3-specific antibody. (C) Functional Ca2+-binding EF-hands are required for CaBP1 to interact with the InsP3R. Endogenous InsP3R-3 from COS-7 cell lysate was pulled down with GST-CaBP1 (wt) but not with GST-CaBP1 triple-EF-hand mutant (mut). Equivalent GST-fusion protein concentrations were present in in vitro binding reactions (Right, Coomassie stain). (D) [Ca2+]-dependence of in vitro interaction of CaBP1 and InsP3R. Endogenous InsP3R-3 in COS-7 cell lysate (1.25 mg) with [Ca2+] fixed as indicated was pulled down with GST-c-CaBP1 and probed with InsP3R-3 antibody. (E) [Ca2+]-dependence of InsP3R-3 interaction with CaBP1 by quantitative densitometry of gels similar to that shown in D (n = 3) with data normalized to binding observed in 500 μM
Figure 3
Figure 3
Typical patch-clamp current records from outer membrane patches obtained from isolated Xenopus oocyte nuclei. Applied potential = 20 mV. The arrows indicate the closed-channel current level. The pipette solutions contained: 10 μM InsP3 (A), 33 nM InsP3 and 1 μM s-CaBP1(B), 33 nM InsP3 (C), 1 μM s-CaBP1 (D), no agonist (E), 1 μM CaBP1 triple-EF-hand mutant (F), 100 nM c-CaBP1 (G), 10 nM c-CaBP1 (H), 1 μM s-CaBP2 (I), 1 μM CaBP5 (J), 12 μM calmodulin (K), or 100 nM c-CaBP1 (L). Current traces D and E, F and G, and K and L (those indicated with braces) were recorded with membrane patches obtained from the same region of the same oocyte nuclei. Free Ca2+ concentrations used in all pipette solutions were optimal for achieving maximum channel Po (1.5–21 μM; ref. 13). n = number of patches used to determine Po. Po = 0.78 ± 0.03, n = 12 (A); Po = 0.65 ± 0.06, n = 4 (B); Po = 0.77 ± 0.08, n = 5 (C); Po = 0.78 ± 0.03, n = 10 (D); Po = 0.71 ± 0.03, n = 9 (G, L); Po = 0.70 ± 0.06, n = 6 (H); Po = 0.87 ± 0.02, n = 7 (I); Po = 0.87 ± 0.02, n = 5 (J). Ca2+. (F) CaBP1-binding affinity for the InsP3R-3. Endogenous InsP3R-3 was pulled down with GST-CaBP1 from COS-7 cell lysates (1.25 mg) containing defined concentrations of s-CaBP1. (G) Quantitative analysis of competition for CaBP1 binding to InsP3R-3 by s-CaBP1 with data normalized to binding in the absence of added s-CaBP1. (H) Specificity of the interaction with the InsP3R of CaBP1 vs. calmodulin (CaM). Endogenous COS-7 cell InsP3R-3 was pulled down with GST-c-CaBP1 from lysates (1.25 mg) supplemented with various concentrations of CaM or s-CaBP1.
Figure 4
Figure 4
Interaction of InsP3R and CaBP1 in brain. (A) Coimmunoprecipitation of CaBP1 with InsP3R-1 (Left and Right) or InsP3R-3 (Left) from whole mouse brain (Left) and cerebellum (Right), but not from nonneural tissues (Left). Immunoprecipitates were probed with anti-CaBP1 antibody. The reciprocal experiment could not be performed because the CaBP antibody is directed against the same region to which the InsP3R binds. (B–F) Confocal immunolocalization of InsP3R-1 and CaBP1 in rat cerebellum sagittal sections. (B) Low magnification. CaBP1 (green) and InsP3R-1 (red) are localized to Purkinje cell somas (PC) and their dendrites in the molecular layer (Mol) (colocalization indicated by yellow). CaBP1 (but not InsP3R-1) is also localized to unidentified fine structures within the granular cell layer (Gr) and to stellate cells (arrow) in the molecular layer. (C–F) Higher magnification demonstrating subcellular colocalization on endoplasmic reticulum. (C) Striated colocalization (yellow) in Purkinje cell primary dendrite. (D–F) Dendritic tips of Purkinje cells. CaBP1 (E; green) and InsP3R-1 (D; red) are colocalized (F; yellow) to linear subregions within thin dendrites (arrowheads).
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