Understanding calmodulin regulation of RyR1 through steady state and stopped-flow fluorescence detection of Ca2+ binding kinetics to calmodulin bound to purified full-length RyR1

Abstract

Calmodulin (CaM) undergoes unique structural changes induced by Ca2+ binding, and thereby activates the skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) at nanomolar [Ca2+] and inhibits it at micromolar [Ca2+]. CaM conversion from RyR1 activator to inhibitor is due to Ca2+ -binding at two EF-hand motifs in each of the two lobes of CaM. However, how CaM’s lobes contribute to this conversion is not fully understood. Here, we introduced single-cysteines for fluorescence labeling in WT and mutant CaMs (Ca2+ binding at N- or C-lobe ablated), attached the environment-sensitive fluorophore acrylodan to either lobe of these CaMs, and monitored fluorescence responses to Ca2+ association/dissociation to CaM free vs.