RT Journal Article
SR Electronic
T1 Functional and Biochemical Properties of Ryanodine Receptor Type 1 Channels from Heterozygous R163C Malignant Hyperthermia-Susceptible Mice
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 420
OP 431
DO 10.1124/mol.110.067959
VO 79
IS 3
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 José R. Lopez
A1 Isaac N. Pessah
YR 2011
UL http://molpharm.aspetjournals.org/content/79/3/420.abstract
AB Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca2+ 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 Ca2+ 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 Ca2+ ([Ca2+]rest) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca2+-ATPase, but RyR1 2844Ser 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 (Po) than WT, with prolonged mean open dwell times and shortened closed dwell times. [3H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with 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 37°C; and 5) elevated cytoplasmic [Ca2+]rest. 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 Ca2+ release unit of adult fibers.