Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Abstract

Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (kd 3 nM, Bmax 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl- and Ca2+, respectively. The reconstituted Ca2+ channels were functionally modified by 1 microM ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca2+ channels had a slope conductance of almost 100 pS in 54 mM CaHEPES and a Ca2+/TRIS+ permeability ratio of 11.0. They also conducted other divalent cations (Ba2+ greater than Ca2+ greater than Sr2+ greater than Mg2+) and were markedly activated by ATP and its nonhydrolysable derivative AMP-PCP (1 mM). Inositol 1,4,5-trisphosphate (1-10 microM) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca2+ channels, sharing some of the characteristics of Ca2+ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca2+ release from internal membrane stores.