Mutation of divergent region 1 alters caffeine and Ca2+ sensitivity of the skeletal muscle Ca2+ release channel (ryanodine receptor)

Abstract

Replacement of amino acids 4187-4628 in the skeletal muscle Ca2+ 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 Ca2+ and to decreased sensitivity of channel inactivation by elevated Ca2+ (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 Ca2+ release and the Ca2+ dependence of [3H]ryanodine binding. Chimera RF10a (amino acids 4187-4381) had a lower EC50 value for activation by caffeine, and RF10c (4557-4628) had a higher EC50 value, whereas the EC50 value for chimera RF10b (43824556) was unchanged. Chimeras RF10b and RF10c were more sensitive to activation by Ca2+, whereas RF10a was less sensitive to inactivation by Ca2+, implicating RF10b and RF10c in Ca2+ activation and RF10a in Ca2+ inactivation. Deletion of much of divergent region 1 sequence to create mutant Δ4274-4535 led to higher caffeine and Ca2+ sensitivity of channel activation and to lower Ca2+ sensitivity for inactivation. Thus, deletion results demonstrate that caffeine, Ca2+, 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 Ca2+ and caffeine sensitivity of the Ca2+ release channel.