Regulation by Ca²⁺ and Inositol 1,4,5-Trisphosphate (Insp₃) of Single Recombinant Type 3 Insp₃ Receptor Channels: Ca²⁺ Activation Uniquely Distinguishes Types 1 and 3 Insp₃ Receptors

The inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) is an endoplasmic reticulum–localized Ca²⁺-release channel that controls complex cytoplasmic Ca²⁺ signaling in many cell types. At least three InsP₃Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP₃R (r-InsP₃R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP₃R-3 exhibited a biphasic dependence on cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP₃R-3 gating was inhibited by high [Ca²⁺]_i with features similar to those of the endogenous Xenopus type 1 InsP₃R (X-InsP₃R-1). Ca²⁺ inhibition of channel gating had an inhibitory Hill coefficient of ∼3 and half-maximal inhibiting [Ca²⁺]_i (K_inh) = 39 μM under saturating (10 μM) cytoplasmic InsP₃ concentrations ([InsP₃]). At [InsP₃] < 100 nM, the r-InsP₃R-3 became more sensitive to Ca²⁺ inhibition, with the InsP₃ concentration dependence of K_inh described by a half-maximal [InsP₃] of 55 nM and a Hill coefficient of ∼4. InsP₃ activated the type 3 channel by tuning the efficacy of Ca²⁺ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP₃R-3 channel was uniquely distinguished from the X-InsP₃R-1 channel by its enhanced Ca²⁺ sensitivity of activation (half-maximal activating [Ca²⁺]_i of 77 nM instead of 190 nM) and lack of cooperativity between Ca²⁺ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP₃R-3 with high gain InsP₃–induced Ca²⁺ release and low gain Ca²⁺–induced Ca²⁺ release properties complementary to those of InsP₃R-1. Thus, distinct Ca²⁺ signals may be conferred by complementary Ca²⁺ activation properties of different InsP₃R isoforms.