Du G G, Khanna V K, MacLennan D H
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 2000 Apr 21;275(16):11778-83. doi: 10.1074/jbc.275.16.11778.
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.
将骨骼肌Ca(2+)释放通道（骨骼肌雷诺丁受体（RyR1））中4187 - 4628位的氨基酸，包括几乎所有的差异区域1（4254 - 4631位氨基酸），替换为相应的心肌雷诺丁受体（RyR2）序列，会导致通道对咖啡因和Ca(2+)激活的敏感性增加，以及对Ca(2+)升高时通道失活的敏感性降低（Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120 - 26126）。在进一步研究中，通过构建新的嵌合体对该区域进行细分，并通过测量体内Ca(2+)释放对咖啡因的依赖性以及[(3)H]雷诺丁结合对Ca(2+)的依赖性来检测通道功能的变化。嵌合体RF10a（4187 - 4381位氨基酸）对咖啡因激活的EC(50)值较低，而RF10c（4557 - 4628位氨基酸）的EC(50)值较高，而嵌合体RF10b（4382 - 4556位氨基酸）的EC(50)值不变。嵌合体RF10b和RF10c对Ca(2+)激活更敏感，而RF10a对Ca(2+)失活较不敏感，这表明RF10b和RF10c参与Ca(2+)激活，而RF10a参与Ca(2+)失活。缺失大部分差异区域1序列以创建突变体Delta4274 - 4535，导致通道激活对咖啡因和Ca(2+)的敏感性更高，而对失活的Ca(2+)敏感性更低。因此，缺失结果表明咖啡因、Ca(2+)和雷诺丁结合位点不在4274 - 4535位氨基酸中。然而，缺失和嵌合突变体的特性表明，RyR1中4274 - 4535位氨基酸以及该区域的三个较短序列（F10a，4187 - 4381位氨基酸；F10b，4382 - 4556位氨基酸；F10c，4557 - 4628位氨基酸）调节Ca(2+)释放通道对Ca(2+)和咖啡因的敏感性。
