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 Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), 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 Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-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.
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Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, 112 College Street, Toronto, Ontario M5G 1L6, Canada.
Oyamada
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Khanna
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MacLennan
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D003408:Cresols / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005334:Fever; D005998:Glycine / Q000737:chemistry*; D015151:Immunoblotting; D007700:Kinetics; D008305:Malignant Hyperthermia / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014162:Transfection
D015220:Calcium Channels; D000697:Central Nervous System Stimulants; D003408:Cresols; D009841:Oligonucleotides; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D000255:Adenosine Triphosphate; C077977:cresol; D002118:Calcium; D005998:Glycine