Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release chan nel (RyR) from rat skeletal muscle was studied under isolated conditions an d in situ. RyRs were either solubilized and incorporated into lipid bilayer s or single fibres were mounted into a Vaseline gap voltage clamp. Single c hannel data were compared to parameters determined from the calculated calc ium release flux. With K+ (250 mM) being the charge carrier the single chan nel conductance was 529 pS at 50 mu M Ca2+ cis and trans, and decreased wit h increasing cis [Ca2+]. Open probability showed a bell shaped calcium depe ndence revealing an activatory and an inhibitory Ca2+ binding site (Hill co efficients of 1.18 and 1.28, respectively) with half activatory and inhibit ory concentrations of 9.4 and 298 mu M. The parameters of the inhibitory si te agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca2+] dependence following a single exponential at every Ca2+ conc entration tested. Closed time histograms, at high [Ca2+], were fitted with three exponentials, from which the longest was calcium independent, and res embled the recovery time constant of SR inactivation (115 +/- 15 ms) obtain ed in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.