Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca2+ release channel (ryanodine receptor) activity and conductance
Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca2+ release channel (ryanodine receptor) activity and conductance
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DOI：
10.1016/s0006-3495(00)76339-9
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发表时间：
2000-08-01
影响因子：
3.4
通讯作者：
Meissner, G
中科院分区：
文献类型：
--
作者：
Gao, L;Balshaw, D;Meissner, G
我们验证了一种假设，即骨骼肌兰尼定受体(RyR1)最大的两个C-末端膜段之间的管腔氨基酸段对通道活动和电导是重要的。获得了11个突变体，并在HEK293细胞中表达，重点是与K+通道选择性过滤器同源的氨基酸残基I4897和M3-M4腔环中的其他6个残基。RyRs和IP(3)Rs(D4903A和D4907A)中不绝对保守的氨基酸突变显示，咖啡因反应细胞内钙释放，钙依赖的[H-3]ryanodine结合，单通道K+和钙电导与野生型RyR1没有显著差异。用I4897对A、L或V或D4917对A进行替换的突变体显示，在完整细胞中，单通道电导降低，高亲和力[H-3]ryanodine结合和钙调节丧失，咖啡因诱导的钙释放发生改变。在RyR和IP3R家族(D4899A、D4899R和R4913E)中氨基酸残基相同的突变通道(D4899A、D4899R和R4913E)显示K+电导降低，失去高亲和力的[H-3]ryanodine结合和失去单通道药理，但在细胞实验中保持其对咖啡因的反应。两个突变体(G4894A和D4899N)在完整细胞和体外都能维持药理调节，但单通道K+和Ca~(2+)电导低于野生型通道。这些结果支持这样的假设，即RyR1最大的两个C末端膜段之间的管腔环区的氨基酸残基构成了RyR1离子传导孔的一部分。
We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid residue I4897 homologous to the selectivity filter of K+ channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not absolutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular Ca2+ release in response to caffeine, Ca2+-dependent [H-3]ryanodine binding, and single-channel K+ and Ca2+ conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [H-3]ryanodine binding and regulation by Ca2+, and an altered caffeine-induced Ca2+ release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP3R families (D4899A, D4899R, and R4913E) exhibited a decreased K+ conductance and showed a loss of high-affinity [H-3]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K+ and Ca2+ conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.