Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inostiol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP(3)R 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 lnsP(3)R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+],), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+], inhibition of lnsP(3)R gating by studying single endogenous type I InsP(3)R 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+], inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] similar to400 nM were inhibited by 290 muM [Ca2+](i), exposure of the isolated nuclei to a bath solution with ultralow [Ca2+] (<5 nM for similar to300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+], up to 1.5 mM. Although lnsP(3) activates gating by relieving high [Ca2+], inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+], inhibition. Our observations suggest that high [Ca2+], 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+], inhibition may not be an immutable property of different lnsP(3)R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the lnsP(3)R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP(3).