Reconstitution of abnormalities in the malignant hyperthermia-susceptible pig ryanodine receptor

Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys⁶¹⁵ ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca²⁺ release channel regulatory properties when compared with normal pigs homozygous for the Arg⁶¹⁵ allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (K(d)) for ryanodine, maximum binding, and Ca²⁺ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca_0.5/²⁺); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had K(d) values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs⁺ conductance, optimal cis-Ca²⁺ for channel opening, and cis-Ca_0.5/²⁺ for channel activation. Significantly, at inactivating levels of cis-Ca²⁺ (>0.1 mM), MHS channels had a greater open probability, a higher cis-Ca_0.5/²⁺ for inhibition of channel opening (250 vs. 75 μM for MHS and normal, respectively), longer mean open times, and shorter mean closed times than did normal channels. We conclude that the mutation at residue 615 causes a detectable alteration in ryanodine receptor/Ca²⁺ channel activity and thus may represent the primary defect responsible for the altered SR Ca²⁺ regulation characteristic of MHS porcine muscle.