Functional and biochemical properties of ryanodine receptor type 1 channels from heterozygous R163C malignant hyperthermia-susceptible mice.

Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca(2+) channel regulation affect skeletal muscle function in myotubes and adult fibers under basal (nontriggering) conditions are not understood. Myotubes, adult flexor digitorum brevis (FDB) fibers, and sarcoplasmic reticulum skeletal membranes were isolated from heterozygous knockin R163C and wild-type (WT) mice. Compared with WT myotubules, R163C myotubes have reduced Ca(2+) transient amplitudes in response to electrical field pulses; however, R163C FDB fibers do not differ in their responses to electrical stimuli, despite heightened cellular cytoplasmic resting Ca(2+) (Ca(2+)) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca(2+)-ATPase, but RyR1 (2844)Ser phosphorylation in R163C muscle is 31% higher than that of WT muscle (p < 0.001). RyR1 channels reconstituted in planar lipid bilayers reveal ∼65% of R163C channels exhibit ≥2-fold greater open probability (P(o)) than WT, with prolonged mean open dwell times and shortened closed dwell times. [(3)H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with WT: 1) 3-fold higher sensitivity to Ca(2+) activation; 2) 2-fold greater [(3)H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic Ca(2+). R163C channels are inherently more active than WT channels, 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 Ca(2+) release unit of adult fibers.