Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+ -release channel (ryanodine receptor isoform 1)

Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca2+-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [H-3]ryanodine binding by Ca2+. caffeine and ATP (adenosine 5'-[beta,gamma -methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca2+ release in HEK-293 cells expressing wildtype and mutant channels. Equilibrium [H-3]ryanodine-binding properties and EC,, values for Ca2+ activation of high-affinity [H-3]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of I mM AMP-PCP, Ca2+-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.