title: Mutation of divergent region 1 alters caffeine and Ca(2+) sensitivity of the skeletal muscle Ca(2+) release channel (ryanodine receptor).
abstract: Replacement of amino acids 4187-4628 in the skeletal muscle Ca(2+) release channel (skeletal ryanodine receptor (RyR1)), including nearly all of divergent region 1 (amino acids 4254-4631), with the corresponding cardiac ryanodine receptor (RyR2) sequence leads to increased sensitivity of channel activation by caffeine and Ca(2+) and to decreased sensitivity of channel inactivation by elevated Ca(2+) (Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120-26126). In further investigations, this region was subdivided by the construction of new chimeras, and alterations in channel function were detected by measurement of the caffeine dependence of in vivo Ca(2+) release and the Ca(2+) dependence of [(3)H]ryanodine binding. Chimera RF10a (amino acids 4187-4381) had a lower EC(50) value for activation by caffeine, and RF10c (4557-4628) had a higher EC(50) value, whereas the EC(50) value for chimera RF10b (4382-4556) was unchanged. Chimeras RF10b and RF10c were more sensitive to activation by Ca(2+), whereas RF10a was less sensitive to inactivation by Ca(2+), implicating RF10b and RF10c in Ca(2+) activation and RF10a in Ca(2+) inactivation. Deletion of much of divergent region 1 sequence to create mutant Delta4274-4535 led to higher caffeine and Ca(2+) sensitivity of channel activation and to lower Ca(2+) sensitivity for inactivation. Thus, deletion results demonstrate that caffeine, Ca(2+), and ryanodine binding sites are not located in amino acids 4274-4535. Nevertheless, the properties of the deletion and chimeric mutants demonstrate that amino acids 4274-4535 and three shorter sequences in this region (F10a, amino acids 4187-4381; F10b, 4382-4556; and F10c, 4557-4628) in RyR1 modulate Ca(2+) and caffeine sensitivity of the Ca(2+) release channel.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
authors.forename: G G
authors.initials: GG
authors.lastname: Du
authors.forename: V K
authors.initials: VK
authors.lastname: Khanna
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D002118:Calcium