The arrhythmogenic calmodulin p.Phe142Leu mutation impairs C-domain Ca2+ binding but not calmodulindependent inhibition of the cardiac ryanodine receptor

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Abstract

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. A number of point mutations in the intracellular Ca 2+ -sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca 2+ binding to CaM and impair inhibition of CaM-regulated Ca 2+ channels like the cardiac Ca 2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca 2+ 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 2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca 2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaMF142L 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 apoCaMF142L 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-F142LCaMBD interaction is structurally different from that of CaM-WT at low Ca 2+ . 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 2+ 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 2+ release by manipulating the CaM-RyR2 interaction.

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Søndergaard, M. T., Liu, Y., Larsen, K. T., Nani, A., Tian, X., Holt, C., … Overgaard, M. T. (2017). The arrhythmogenic calmodulin p.Phe142Leu mutation impairs C-domain Ca2+ binding but not calmodulindependent inhibition of the cardiac ryanodine receptor. Journal of Biological Chemistry, 292(4), 1385–1395. https://doi.org/10.1074/jbc.M116.766253

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