Mechanism of Calmodulin Inhibition of Cardiac Sarcoplasmic Reticulum Ca 2+ Release Channel (Ryanodine Receptor)

Abstract

The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca 2+ release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca 2+ concentrations. CaM reduced channel open probability at <10 μM Ca 2+ by decreasing channel events and mean open times and increasing mean close times. At >10 μM Ca 2+, CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to −50 mV and from −50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca 2+ fluxes. CaM decreased the duration of the open events after the voltage switch from −50 mV to +50 mV. In parallel experiments, a Ca 2+-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca 2+ release.