A number of point mutations in the intracellular Ca-sensing protein calmodulin (CaM) are arrhythmogenic， yet their underlying mechanisms are not clear. These mutations generally decrease Ca binding to CaM and impair inhibition of CaM-regulated Ca channels like the cardiac Ca release channel (ryanodine receptor， RyR2)， and it appears that attenuated CaM Ca binding correlates with impaired CaM-dependent RyR2 inhibition. Here， we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met)， which markedly reduces CaM Ca binding. Surprisingly， CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca release in HEK293 cells... More
A number of point mutations in the intracellular Ca-sensing protein calmodulin (CaM) are arrhythmogenic， yet their underlying mechanisms are not clear. These mutations generally decrease Ca binding to CaM and impair inhibition of CaM-regulated Ca channels like the cardiac Ca release channel (ryanodine receptor， RyR2)， and it appears that attenuated CaM Ca binding correlates with impaired CaM-dependent RyR2 inhibition. Here， we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met)， which markedly reduces CaM Ca binding. Surprisingly， CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca release in HEK293 cells compared with CaM-WT. Furthermore， CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I， D96V， N98S， and D130G， which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover， NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD， which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L， despite its reduced Ca binding. Collectively， these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca release by manipulating the CaM-RyR2 interaction.
