Regulation of the RYR1 and RYR2 Ca2+ Release Channel Isoforms by Ca2+-Insensitive Mutants of Calmodulin

Abstract

Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca²⁺; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca²⁺ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca²⁺, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca²⁺, RYR1 inhibition by Ca²⁺CaM was abolished by mutations targeting CaM's C-terminal Ca²⁺ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca²⁺ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca²⁺ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca²⁺ EC₅₀ increased 7−10-fold in the presence of 5 μM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca²⁺ sites. Furthermore, a mutant CaM deficient in Ca²⁺ binding at all four Ca²⁺ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca²⁺CaM. We conclude that Ca²⁺ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca²⁺ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.