ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating

Abstract

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.

Figure 1

Ca²⁺ dependencies of types 1 and 3 InsP₃R channel P _o in the presence of 0.5 mM ATP. 10 μM InsP₃ was present in the pipet solutions used in experiments presented in all figures. Open triangles represent data for X-InsP₃R-1 obtained from uninjected oocytes. Closed squares represent data for r-InsP₃R-3 obtained from cRNA-injected oocytes. The curves (dashed for X-InsP₃R-1 and solid for r-InsP₃R-3) are the biphasic Hill equation fits from Mak et al. 2001.

Figure 2

(A–D) Typical single-channel current traces of r-InsP₃R-3 channels in the outer membrane of nuclei isolated from r-InsP₃R-3 cRNA-injected oocytes under suboptimal [Ca²⁺]_i, with various [ATP]_i and [Mg²⁺]_i. Arrows indicate closed channel current levels. (A) [Ca²⁺]_i = 224 nM, [ATP]_i = 0.5 mM; [Mg²⁺]_i = 0 mM. (B) [Ca²⁺]_i = 255 nM, [ATP]_i = 0 mM; [Mg²⁺]_i = 0 mM. (C) [Ca²⁺]_i = 274 nM, [ATP]_i = 0 mM; [Mg²⁺]_i = 3.0 mM. (D) [Ca²⁺]_i = 286 nM, [ATP]_i = 12 μM; [Mg²⁺]_i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg²⁺). Reduction of InsP₃R channel conductance in the presence of Mg²⁺ (C and D) is caused by permeant ion block of the channel by the divalent cation (Mak and Foskett 1998). (E) Single-channel P _o of the r-InsP₃R-3 channel in conditions specified in A–D. Asterisk represents P < 0.05.

Figure 3

(A–D) Typical single-channel current traces of r-InsP₃R-3 channels under optimal [Ca²⁺]_i with various [ATP]_i and [Mg²⁺]_i. Arrows indicate closed channel current levels. (A) [Ca²⁺]_i = 4.4 μM, [ATP]_i = 0.5 mM; [Mg²⁺]_i = 0 mM. (B) [Ca²⁺]_i = 5.6 μM, [ATP]_i = 0 mM; [Mg²⁺]_i = 0 mM. (C) [Ca²⁺]_i = 2.5 μM, [ATP]_i = 0 mM; [Mg²⁺]_i = 3.0 mM. (D) [Ca²⁺]_i = 2.5 μM, [ATP]_i = 12 μM; [Mg²⁺]_i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg²⁺). Reduction of InsP₃R channel conductance in the presence of Mg²⁺ (C and D) is caused by permeant ion block of the channel by the divalent cation (Mak and Foskett 1998). (E) Single-channel open probability (P _o) of the r-InsP₃R-3 channel in conditions specified in A–D.

Figure 4

(A) Ca²⁺ dependencies of the r-InsP₃R-3 channel P _o in the presence of various [ATP]_i. The symbols correspond to P _o data in various [ATP]_i (in millimolars) as tabulated in the graph. The curves for 0 and 0.5 mM ATP represent theoretical fits to the data using the biphasic Hill equation () whereas the curve for 0.3 mM ATP represents a fit using the activation Hill equation (). Hill equation parameters used are tabulated in Table . (B–C) Typical single-channel current traces of r-InsP₃R-3 channels under inhibiting [Ca²⁺]_i. Arrows indicate closed channel current levels. (B) [Ca²⁺]_i = 58 μM, [ATP]_i = 0.5 mM; [Mg²⁺]_i = 0 mM. (C) [Ca²⁺]_i = 83 μM, [ATP]_i = 0 mM; [Mg²⁺]_i = 0 mM.

Figure 5

Ca²⁺ dependencies of types 1 and 3 InsP₃R channel P _o in the absence of ATP. Open triangles represent data for X-InsP₃R-1 obtained from uninjected oocytes. Closed squares represent data for r-InsP₃R-3 obtained from cRNA-injected oocytes. The curves (dashed for X-InsP₃R-1 and solid for r-InsP₃R-3) are the biphasic Hill equation fits using and parameters tabulated in Table .

Figure 6

Ca²⁺ dependencies of the mean open and closed channel durations of the r-InsP₃R-3 channels in the presence of 0 (▪) or 0.5 mM (○) cytoplasmic free ATP. In the closed channel duration graph, data points obtained with the same InsP₃ concentrations are connected with a line for clarity.

Figure 7

Variation of the ratio between the P _o of r-InsP₃R-3 (₃ P _o) and that of X–InsP₃R-1 (₁ P _o), with [Ca²⁺]_i in the presence of different [ATP]_i as tabulated. P _o are calculated with , using the parameters (P _max, K _act, and H _act) tabulated in Table . ₃ P _o for 4.8 and 9.5 mM ATP were calculated using the same parameters as in 0.5 mM ATP. P _max of X-InsP₃R-1 was assumed to be 0.8 in 0.3 mM ATP.