Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor Calcium-release Channel

The inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP ₃R), a Ca²⁺-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca ²⁺ signals in many cell types. Three InsP₃R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP₃R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]_i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca²⁺] in mediating high [Ca²⁺] _i inhibition of InsP₃R gating by studying single endogenous type 1 InsP₃R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca²⁺-insensitive mutant calmodulin affected inhibition of gating by high [Ca²⁺]_i. However, a novel, calmodulin-independent regulation of [Ca²⁺]_i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca²⁺] ∼400 nM were inhibited by 290 μM [Ca²⁺]_i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca²⁺] (<5 nM, for ∼300 s) before the patch-clamp experiments reversibly relieved Ca²⁺ inhibition, with channel activities observed in [Ca²⁺]_i up to 1.5 mM. Although InsP₃ activates gating by relieving high [Ca²⁺]_i inhibition, it was nevertheless still required to activate channels that lacked high [Ca²⁺]_i inhibition. Our observations suggest that high [Ca²⁺]_i inhibition of InsP₃R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca²⁺]_i inhibition may not be an immutable property of different InsP₃R isoforms. Furthermore, these observations support an allosteric model in which Ca²⁺ inhibition of the InsP₃R is mediated by two Ca ²⁺ binding sites, only one of which is sensitive to InsP₃.