Unitary Calcium Current Through Recombinant Mammalian Type 3 IP3 Receptor Channels Under Physiological Ionic Conditions

Abstract

The ubiquitous inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel, localized primarily in the ER membrane, releases Ca2+ into the cytoplasm upon binding IP3, generating and modulating intracellular Ca2+ signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability (Po) of the active channels, the size of the unitary Ca2+ current (iCa) passing through an open IP3R channel determines the amount of Ca2+ released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca2+ signals generated in response to extracellular stimuli. Despite its significance, iCa for IP3R channels in physiological ionic conditions has not been directly measured. Here we report the first measurement of iCa through an IP3R channel in its native membrane environment under physiological ionic conditions. Nuclear patch-clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 IP3R channels. Within physiological ranges of free Ca2+ concentrations in the ER lumen ([Ca2+]ER), free cytoplasmic [Ca2+] ([Ca2+]i) and symmetric free [Mg2+] ([Mg2+]f), the iCa-[Ca2+]ER relation was linear with no detectable dependence on [Mg2+]f. iCa was 0.15 ± 0.01 pA for a filled ER store with 500 μM [Ca2+]ER. The iCa-[Ca2+]ER relation suggests that Ca2+ released by an IP3R channel raises [Ca2+]i near the open channel to ∼ 13-70 μM, depending on [Ca2+]ER. These measurements have implications for the activities of nearby IP3-liganded IP3R channels, and they confirm that Ca2+ released by an open IP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release.