Malignant Hyperthermia Susceptibility Mutation Cav1.1 R174W Dramatically Alters RyR1 Single Channel Function

Abstract

Nearly 200 variants in the gene encoding the skeletal muscle RyR1 Ca2+ channel are associated with MHS but only 30 were demonstrated to perturb channel function. We previously showed that mouse MHS mutations RyR1_R163C or RyR1_T4826I have abnormally high open probabilities and elevated [3H]ryanodine binding when compared to wild type. Recently, a small number of MHS families were shown to express mutations, not in the RyR1, but rather in the gene encoding the skeletal muscle L-type Ca2+ channel (CaV1.1). We therefore generated a knock-in mouse line expressing CaV1.1_R174W that confers MHS in human kindred. Homozygous (HOM) mice survive, but trigger with fulminant MH when exposed to halogenated volatile anaesthetics, have chronically elevated resting myoplasmic Ca2+, and lack L-type Ca2+ current in adult Flexor digitorum brevis fibers (Bannister et al, this meeting). It is unknown however if MHS mutations residing within CaV1.1 are capable of modifying RyR1 structure/function thereby contributing to disrupted intracellular Ca2+ homeostasis. To address this question, SR membranes were prepared from the skeletal muscles of age-matched WT and HOM Cav1.1_R174W for biochemical and electrophysiological analysis. Unexpectedly, we found significantly abnormal properties of HOM as compared to WT in: (1) enhanced [3H]Ry binding; (2) elevated levels of RyR1/FKBP12 expression, and (3) exceptionally higher Po with channels in BLM at all [Ca2+]cis. Even at [Ca2+]cis=120nM, an inhibitory condition to RyR1 in normal WT muscle fibers, RyR1 channels from CaV1.1_R174W muscle maintained a conformation accessible for ryanodine binding. These results demonstrate that a MHS mutation residing outside of RyR1 is capable of persistently modifying RyR1 channel function. These findings have significant implications about the molecular mechanisms that lead to MHS and fulminant MH. (Grant supports: AR052534 to PDA, KGB, PMH, CFA, INP; AR055104 and MDA277475 to KGB).