EFFECTS OF K+ ON THE BINDING OF CA2+ TO THE CA2+-ATPASE OF SARCOPLASMIC-RETICULUM

Equilibrium and kinetic fluorescence methods have been used to characterize the interactions between K+ and the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum. K+ shifts the E2-E1 equilibrium of the ATPase towards E1 and increases the rate of Ca2+ binding to the ATPase, as detected by changes in tryptophan fluorescence intensity, suggesting that K+ increases the rate of the E2-E1 transition. The data are consistent with binding of K+ at the inner Ca2+-binding site on the ATPase in competition with H+ and Mg2+, with a higher affinity in the E1 than in the E2 conformation. K+ has no effect on the affinity for Mg2+, as detected by changes in tryptophan fluorescence intensity; since it has been proposed that the changes in tryptophan fluorescence follow from binding to Mg2+ at the outer Ca2+-binding site, this suggests that K+ is unable to bind at the outer Ca2+-binding site. K+ increases the rate of dissociation of Ca2+ from the Ca2+-bound ATPase and reduces the effect of Mg2+ On the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It is suggested that these effects of K+ are the result of binding at a 'gating' site on the ATPase, in competition with binding of H+. Binding of K+ at the inner Ca2+ binding site and at the gating site account for the observed effects of K+ on the affinity of the ATPase for Ca2+.