RT Journal Article SR Electronic T1 Functional and Biochemical Properties of RyR1 Channels from Heterozygous R163C Malignant Hyperthermia Susceptible Mice JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.110.067959 DO 10.1124/mol.110.067959 A1 Wei Feng A1 Genaro C. Barrientos A1 Gennady Cherednichenko A1 Tianzhong Yang A1 Isela T. Padilla A1 Kim Truong A1 Paul D. Allen A1 Jose R. Lopez A1 Isaac N. Pessah YR 2010 UL http://molpharm.aspetjournals.org/content/early/2010/12/14/mol.110.067959.abstract AB 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.