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.

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 P_o (1.5–21 μM; ref. 13). n = number of patches used to determine P_o. P_o = 0.78 ± 0.03, n = 12 (A); P_o = 0.65 ± 0.06, n = 4 (B); P_o = 0.77 ± 0.08, n = 5 (C); P_o = 0.78 ± 0.03, n = 10 (D); P_o = 0.71 ± 0.03, n = 9 (G, L); P_o = 0.70 ± 0.06, n = 6 (H); P_o = 0.87 ± 0.02, n = 7 (I); P_o = 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.

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

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).