Graded recruitment and inactivation of single lnsP_3 receptor Ca~(2+)-release channels: implications for quartal Ca~(2+)release

摘要

Modulation of cytoplasmic free Ca~(2+) concentration ([Ca~(2+)]i) by receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP_3) and activation of its receptor (InsP_3R), a Ca~(2+)-release channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP_3-mediated signalling is the graded release of Ca~(2+) in response to incremental levels of stimuli. Ca~(2+) release has a transient fast phase, whose rate is proportional to [InsP_3], followed by a much slower one even in constant [InsP_3]. Many schemes have been proposed to account for quantal Ca~(2+) release, including the presence of heterogeneous channels and Ca~(2+) stores with various mechanisms of release termination. Here, we demonstrate that mechanisms intrinsic to the single InsP_3R channel can account for quantal Ca~(2+) release. Patch-clamp electrophysiology of isolated insect Sf9 cell nuclei revealed a consistent and high probability of detecting functional endogenous InsP_3R channels, enabling InsP_3-induced channel inactivation to be identified as an inevitable consequence of activation, and allowing the average number of activated channels in the membrane patch (N_A) to be accurately quantified. InsP_3-activated channels invariably inactivated, with average duration of channel activity reduced by high [Ca~(2+)]_i and suboptimal [InsP_3]. Unexpectedly, N_A was found to be a graded function of both [Ca~(2+)]_i and [InsP_3]. A qualitative model involving Ca~(2+)-induced InsP_3 R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand sensitivity can be generated in a homogeneous population of InsP_3R channels, providing a mechanism for graded Ca~(2+) release that is intrinsic to the InsP_3R Ca~(2+) release channel itself.