TY - JOUR T1 - Functional and Biochemical Properties of RyR1 Channels from Heterozygous R163C Malignant Hyperthermia Susceptible Mice JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.110.067959 SP - mol.110.067959 AU - Wei Feng AU - Genaro C. Barrientos AU - Gennady Cherednichenko AU - Tianzhong Yang AU - Isela T. Padilla AU - Kim Truong AU - Paul D. Allen AU - Jose R. Lopez AU - Isaac N. Pessah Y1 - 2010/12/14 UR - http://molpharm.aspetjournals.org/content/early/2010/12/14/mol.110.067959.abstract N2 - Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility (MHS). How inherent impairments in Ca2+ channel regulation impact skeletal muscle function in myotubes and adult fibers under basal (non-triggering) conditions are not understood. Myotubes, adult FDB fibers, and SR skeletal membranes were isolated from heterozygous knock-in R163C and wild type (WT) mice. Compared to WT, R163C myotubes have reduced Ca2+ transient amplitudes in response to electrical field pulses, however R163C adult fibers do not differ in their responses to electrical stimuli, despite heightened [Ca2+]rest and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FKBP12 and Ca2+-ATPase expression, but RyR1 2844Ser phosphorylation in R163C muscle is ~31% higher than WT (p<0.001). RyR1 channels reconstituted in planar lipid bilayers (BLM) reveal ~65% of R163C channels exhibit >2-fold greater open probability (Po) than WT, with prolonged mean open-dwell times and shortened closed-dwell times. [3H]Ry binding and single channel analyses show that R163C-RyR1 has altered regulation compared to WT: (1) 3-Fold higher sensitivity to Ca2+ activation; (2) 2-Fold greater [3H]Ry receptor occupancy; (3) comparatively higher channel activity, even in reducing glutathione buffer; (4) enhanced RyR1 activity both at 25 and 37oC; (5) Elevated cytoplasmic [Ca2+]rest. R163C channels are inherently more active than WT, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the CRU of adult fibers. ER -