Calcium Spark Termination: Ryanodine Receptor Unitary Flux Dependent Mechanism

Abstract

Spontaneous sparks seem to terminate at a fixed free sarcoplasmic recticulum Ca concentration ([Ca]SR) indicating that the SR luminal Ca level is a key factor in terminating Ca sparks. In principle, such luminal Ca control could be achieved by different mechanisms. One is that luminal Ca may alter RyR2 gating by acting at intra-SR sites. Another is that, as luminal Ca falls, RyR2 unitary release flux (iCa) may become insufficient to support continued inter-RyR2 Ca-induced Ca release within a RyR2 cluster (a cytosolic process). To date, it has been virtually impossible to experimentally distinguish these possibilities in cells. We have overcome this obstacle by devising a means to manipulate iCa independently of [Ca]SR. This was accomplished by exploiting RyR2 permeation properties. Briefly, sparks and [Ca]SR were simultaneously recorded in permeabilized rat myocytes. Unitary RyR2 iCa in the tested cellular solutions was defined using single RyR2 measurements in bilayers as well as a well-established RyR permeation model. Preliminary data reveal that reducing RyR2 iCa (at a relatively constant [Ca]SR) dramatically decreases spark frequency. We believe this method is the first to experimentally delineate the contribution of RyR2 iCa flux in the SR luminal Ca control of sparks in cardiomyocytes.