Inositol trisphosphate receptor Ca2+ release channels

Abstract

The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R.

FIG. 1

Schematic of the behaviors of inositol trisphosphate receptor (InsP₃R) channels in the presence of increasing concentrations of InsP₃. InsP₃Rs are shown arranged in clusters that form discrete release sites within the continuous endoplasmic reticulum. A: at low [InsP₃] during weak agonist stimulation, few receptors (in green) bind InsP₃. Others (in yellow) are not InsP₃ liganded and therefore not activated. Consequently, highly localized small Ca²⁺ signals (“blips”) are generated by Ca²⁺ released through a single or few InsP₃R channels raising cytoplasmic Ca²⁺ concentration (shown in red). B: at higher levels of [InsP₃], coordinated openings of several channels (InsP₃ liganded) within a cluster is triggered by Ca²⁺ release from one channel acting as an activating ligand to stimulate gating of nearby channels through a process of Ca²⁺-induced Ca²⁺ release (CICR). C: even higher [InsP₃] evokes global propagating Ca²⁺ signals (waves). Ca²⁺ released at one cluster can trigger Ca²⁺ release at adjacent clusters by CICR, leading to the generation of Ca²⁺ waves that propagate by successive cycles of Ca²⁺ release, diffusion, and CICR. [Figure kindly supplied by I. Parker and N. Callamaras. Adapted from Parker et al. (358).]

FIG. 2

Structural determinants of the InsP₃R. A: overall domain structure. The InsP₃R molecule depicted as a linear amino acid sequence, with the NH₂-terminal InsP₃ binding region (red), coupling region (yellow), transmembrane region (green), and COOH tail (blue) depicted. B: linear amino acid sequence. Residues are numbered according to the rat type 1 SI+, SII+, SIII− sequence (protein accession no. 121838). Structural features (see section in this review where each element is described) shown are as follows: arm subdomain and β-trefoil in the InsP₃-binding suppressor domain (sect. IIIB1); β-trefoil and armadillo repeats in InsP₃ binding core domain (sect. IIIB1); armadillo repeats in the coupling domain (sect. IIIB3); alternative splicing regions SI, SII, and SIII (sect. IIB2) for type 1 InsP₃R; opt deletion in type 1 InsP₃R mutant (sect. IIIB3); ATP-binding sites ATPA, ATPB, and ATPC (sect. VIF4); transmembrane helices TM1–6 and pore-forming P region (sect. IIIB2A) with selectivity filter (sect. VF); linker region (sect. IIIB1); dimerizing region (sect. IIIC1); tetramer forming region (sect. IIIC1). Trypsin proteolysis sites (sect. IIIC3) are indicated by black arrowheads. The caspase 3 cleavage site (sect. IIIC3) is also shown. G25 (sect. IIIB1), S217 (sect. IIIB1), T799 (sect. IIIB3), M837 (sect. IIIB3), C1430 (sect. IIIB3), and G2045 (sect. IIIB3) are highly conserved residues the mutation of which can impact InsP₃R channel functions. Mutation of E2100 modifies Ca²⁺ regulation of InsP₃R channel (sect. VIK). R265, L508, and R511 are critically important for InsP₃ binding (sect. IIIB1). G2586, T2591, L2592, and F2595 are residues that may be involved in forming the gate of the InsP₃R channel (sect. IIIB2B). N2475 and N2503 are glycosylation sites (sect. IIIB). S1589 and S1755 are PKA/PKG phosphorylation sites (sect. VIL, 1 and 2); S2681 is an Akt phosphorylation site (sect. VIL4); Y353 is a Fyn tyrosine kinase phosphorylation site (sect. VIL5); and S421 and T799 are cdc2/CyB phosphorylation sites (sect. VIL6). Sequences involved in interaction of InsP₃R channel with the following proteins are also depicted: homer (sect. VIN13); calmodulin (sect. VIN1); CaBP (sect. VIN2); RACK1 (sect. VIN3); IRBIT (sect. VIN4); CIB1 (sect. VIN2); Na⁺-K⁺-ATPase (sect. VIN14); COOH terminal of InsP₃R in the tetrameric channel (sect. IIIB1); TRPC3 (sect. VIN14); GAPDH (sect. VIN8); AKAP9 [through leucine/isoleucine zipper (LIZ) motif] (sect. VIL1); FKBP12 (sect. VIN7); Erp44 (sect. VIN6); chromogranins (sect. VIN5); cytochrome c (sect. VIN10); HAP1 and Htt^exp (sect. VIN11); protein 4.1N (sect. VIN13); and PP1 (sect. VIL1).

FIG. 3

The InsP₃R Ca²⁺ release channel. Cartoon depicting three of four InsP₃R molecules (in different colors) in a single tetrameric channel structure. Part of the luminal loop connecting transmembrane helices 5 and 6 of each monomer dips into the fourfold symmetrical axis, creating the permeation pathway for Ca²⁺ efflux from the lumen of the endoplasmic reticulum.

FIG. 4

Structures of the InsP₃R. A: crystal structures of the core InsP₃ binding domain (left) and suppressor domain (right). InsP₃ present in the core domain structure coordinated in a cleft created by an NH₂-terminal β-sheet-rich β-trefoil domain and an α-helical armadillo-repeat domain. Suppressor domain is comprised entirely of a β-trefoil domain (head) with a helical insert (arm). Structures solved in Refs. , . B: cryoelectron microscopic single-particle reconstruction of InsP₃R (right, tilted with respect to the plane of the page with cytoplasmic aspect facing upwards toward viewer with InsP₃ binding core domain density fitted into an L-shaped density). For a better fit, various parts of the InsP₃ binding core domain were rotated as indicated by the arrows with respect to the crystal structure shown in A. N and C refer to NH₂ and COOH termini of each domain in A and B. [From Sato et al. (407), with permission from Elsevier.]

FIG. 5

Nuclear patch-clamp electrophysiology. A: schematic of cell nucleus, illustrating that the outer membrane of the double-membrane nuclear envelope is continuous with the endoplasmic reticulum (ER), with the lumen between the two membranes continuous with the ER lumen. Patch-clamping isolated Xenopus oocyte nucleus (B) and insect Sf9 cell nucleus (C) visualized on the stage of a patch-clamp microscope, with patch pipettes forming giga-ohm seals on the outer nuclear membrane. Horizontal shadow over the Xenopus nucleus is the edge of a stabilizing piece of coverslip. Intact Sf9 cell is also present in C. [B modified from Mak et al. (287).]

FIG. 6

Typical single-channel current traces of X-InsP₃R-1 in various cytoplasmic Ca²⁺ concentrations and saturating 10 μM InsP₃. Current traces were recorded during nuclear patch-clamp experiments at cytoplasmic Ca²⁺ concentrations as tabulated, in 0.5 mM free ATP. All current traces in this and other graphs were recorded at 20 mV. Arrows indicate closed-channel current level in all current traces. Channel open probability (P_o) was evaluated for the single-channel patch-clamp experiments yielding the current traces shown in A, B, C, and D of 0.008, 0.50, 0.89, and 0.002, respectively. [Modified from Mak et al. (282).]

FIG. 7

[Ca²⁺]_i and [InsP₃] regulation of InsP₃R channel activity. A: [Ca²⁺]_i dependence of mean P_o of endogenous X-InsP₃R-1 channels (solid symbols) in various [InsP₃] as tabulated. [Modified from Mak et al. (282).] Each data point in this and subsequent P_o versus [Ca²⁺ ]_i plots is the average of channel P_o from at least 4 experiments using the same ligand concentrations. The curves are least-squares fit of the data points using the biphasic Ca²⁺ regulation Hill equation (Eq. 1) with parameters as tabulated. The large open circles represent P_o for recombinant rat InsP₃R-1 channels in various [Ca²⁺]_i in saturating 10 μM InsP₃. [Modified from Boehning et al. (42).] Inset: plot of K_inh derived from the biphasic Hill equation fit of P_o data versus [InsP₃] used. The curve is the least-squares fit of the K_inh values using the activation Hill equation $K_{\text{inh}}=K_{\text{inh}}^{\infty }{\{1+{({}_{\text{inh}}{}^{{\text{IP}}_3}K/[{\text{InsP}}_3])}^{{}_{\text{inh}}{}^{{\text{IP}}_3}H}\}}^{-1}$ with parameters as tabulated. B: [Ca²⁺]_i dependence of mean P_o of recombinant r-InsP₃R-3 channels in various [InsP₃] as tabulated. [Modified from Mak et al. (283).] Data points and fitted curves are obtained as described for A. C: [Ca²⁺]_i dependence of mean P_o of endogenous InsP₃R channels from Sf9 cells in various [InsP₃] as tabulated. [Modified from Ionescu et al. (196).] Data points, fitted curves, and inset graph are obtained as described for A. D: [Ca²⁺]_i dependence of mean P_o of X-InsP₃R-1 InsP₃R channels that have been exposed to bath solution with very low [Ca²⁺]_i (< 5 nM) for a few minutes before the patch-clamp experiments, in various [InsP₃] as tabulated. The curves are least-squares fits to the data using activation Hill equation P_o = P_Hill{1 + (K_act/[Ca²⁺]_i)^H_act}⁻¹ with parameters as tabulated. [Modified from Mak et al. (286).]

FIG. 8

[Ca²⁺]_i dependence of vertebrate InsP₃R channel activity in saturating [InsP₃] observed in various single-channel studies. Biphasic Hill equation curves shown are generated either using parameters provided by studies cited below, or by fitting data provided in those studies with the Hill equation. Entries denoted by red letters are data from endogenously expressed InsP₃R channels; other entries denoted by black letters are from recombinant homotetrameric InsP₃R channels. Entries marked with asterisks are obtained by nuclear patch-clamp experiments; others are from InsP₃R channels reconstituted into planar lipid bilayers. All data were observed in the presence of 0.5–1 mM Na₂ ATP on the cytoplasmic side of the channel unless stated otherwise. A: canine cerebellar (438). B: bovine cerebellar in 0 ATP (382). C: rat type 1 SI+ SII+ in COS cells in 0 ATP (381). D: rat cerebellar (478). E: rat type 1 SI− SII+ in Sf9 cells (478). F: rat type 1 SI− SII+ in Sf9 cells (479). G: Xenopus oocyte (282). H: rat cerebellar (294). I: ferret cardiac ventricular myocyte in 0 ATP (382). J: rat type 2 in COS cells in 0 ATP (380). K: rat type 2 in Sf9 cells (481). L: rat pancreatic RIN-m5F cells (163). M: rat type 3 in Sf9 cells in 5 mM Na₂ATP (481). N: rat type 3 in Xenopus oocytes (283).

FIG. 9

Biphasic Hill equation fits to InsP₃R channel P_o versus [Ca²⁺]_i data. Open squares are experimental data, and smooth curves are Hill equation fit to the data using parameters as tabulated. A: channel P_o data for recombinant rat type 1 E2100D mutant InsP₃R expressed in Sf9 cells were fitted (black curve) using modified biphasic Hill equation (Eq. 2) with parameters given in Ref. , tabulated in black. An alternative Hill equation fit (thick yellow curve) using the same equation with a different set of parameters (tabulated in yellow) is effectively indistinguishable from the fit provided in Ref. . B: channel P_o data for recombinant wild-type Drosophila InsP₃R channel expressed in Sf9 cells were fitted (black curve) using modified biphasic Hill equation (Eq. 2) with parameters given in Ref. , tabulated in black. The more general Hill equation (Eq. 1) gives a better fit to the data (red curve) using parameters tabulated in red.

FIG. 10

Regulation of InsP₃R channel activity by ATP. A: [Ca²⁺]_i dependence of mean P_o of endogenous X-InsP₃R-1 channels in the presence of various [ATP]_free as tabulated. [Modified from Mak et al. (281).] Solid symbols represent data obtained in the absence of Mg²⁺. Open circles represent data obtained in 3 mM Mg²⁺ and 0 ATP. Open squares represent data obtained in 3 mM Mg²⁺ and 0.5 mM total [ATP], with [ATP]_free = 12 μM calculated by MaxChelator. The curves are fits using either the biphasic Hill equation (Eq. 1) to the data in [ATP]_free = 0 (blue) or 0.5 mM (black); or the activating Hill equation (see legend for Fig. 7) to the data in other [ATP]_free (10 nM < [Ca²⁺]_i < 1 μM). Biphasic Hill equation parameters fitting data in 0 [ATP]_free (blue curve) are tabulated. Inset: plot of the Hill equation parameter K_act versus [ATP]_free used. The curve is a fit to the K_act values using the modified Michaelis-Menten equation $K_{\text{act}}=K_{\text{act}}^{0\text{ATP}}+(K_{\text{act}}^{\infty \text{ATP}}-K_{\text{act}}^{0\text{ATP}}){\{1+({[\text{ATP}]}_{\text{free}}/{}_{\text{act}}{}^{\text{ATP}}K)\}}^{-1}$ with parameters as tabulated. B: [Ca²⁺]_i dependence of mean P_o of recombinant r-InsP₃R-3 channels in the presence of various [ATP]_free as tabulated. [Modified from Mak et al. (280).] Solid symbols, open circles, and open squares represent data in a similar convention as described for A. Solid curves are either biphasic Hill equation fit or activation Hill equation fit to the data in various [ATP]_free as described for A. The thick dashed blue curve is the biphasic Hill equation fit to channel P_o for X-InsP₃R-1 in the absence of ATP plotted for comparison. Parameters are tabulated for biphasic Hill equation fit to r-InsP₃R-3 channel P_o in 0 ATP (in blue), as well as those for activating Hill equation fit to r-InsP₃R-3 channel P_o in [ATP]_free = 0.3 mM (in yellow) and 0.5 mM (in black) ([Ca²⁺]_i < 1 μM). C: [Ca²⁺]_i dependence of mean P_o of endogenous X-InsP₃R-1 channels in 0 ATP in the presence of various [InsP₃] as tabulated. The curves are fits to the data using biphasic Hill equation (Eq. 1) with parameters as tabulated. [Modified from Mak et al. (279).] D: channel P_o versus [ATP]_free curves calculated for X-InsP₃R-1 channels in 10 μM InsP₃ and various [Ca²⁺]_i as labeled, using the biphasic Hill equation (Eq. 1) and the modified Michaelis-Menten equation in A.

FIG. 11

Typical single-channel current traces of X-InsP₃R-1 in various [Mg²⁺] and [ATP]_free. A: current trace was recorded at optimal (6.2 μM) [Ca²⁺]_i and saturating (10 μM) [InsP₃] in the absence of ATP and Mg²⁺.[Modified from Mak et al. (279).] The remaining current traces were recorded at 0.25 μM [Ca²⁺]_i and saturating [InsP₃] (10 μM). [Modified from Mak et al. (281).] B: [ATP]_free = 0.5 mM, [Mg²⁺] = 0 mM. C: [ATP]_free = 0 mM, [Mg²⁺] = 0 mM. D: [ATP]_free = 0 mM, [Mg²⁺] = 3 mM. E: total [ATP] = 0.5 mM, [Mg²⁺] = 3 mM, [ATP]_free = 12 μM calculated by MaxChelator software (29). F: total [ATP] = 4.8 mM, [Mg²⁺] = 3 mM, [ATP]_free = 1.9 mM calculated by MaxChelator. Arrows indicate closed-channel current level in the traces. Channel P_o evaluated for the single-channel patch-clamp experiments yielding the current traces shown in A–F are 0.79, 0.46, 0.10, 0.10, 0.10, and 0.76, respectively.

FIG. 12

InsP₃R channel activities activated by adenophostin A (AdA). [Ca²⁺]_i dependence of mean P_o of endogenous X-InsP₃R-1 channels in the presence of various ligand (AdA or InsP₃) concentrations as tabulated in 0.5 mM [ATP]_free (A) or 0 [ATP]_free (B). The curves are biphasic Hill equation (Eq. 2) fit to the data using parameters as tabulated. Typical single-channel current traces of X-InsP₃R-1 in outer nuclear membrane recorded at optimal [Ca²⁺]_i and saturating 100 nM AdA in 0.5 mM [ATP]_free with channel P_o of 0.72 (C) and 0 [ATP]_free with channel P_o of 0.39 (D). [Modified from Mak et al. (279).]

FIG. 13

Dependencies on [Ca²⁺]_i and [InsP₃] of InsP₃R channel activity duration and recruitment. A: channel activity duration. Data points are averages of channel activity durations in ligand conditions as tabulated. Smooth curves in graphs in this figure were drawn by hand for clarity. B: ligand-dependent recruitment of InsP₃R. Data points are average number of active channels in membrane patches (N_A) in ligand concentrations as tabulated. C: ligand-dependent relative magnitude of InsP₃R-mediated Ca²⁺ release. The product N_AP_o, determined using data shown in Fig. 6B and Fig. 1D, in ligand concentrations as tabulated. [Modified from Ionescu et al. (196).]