Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina , and Cardiac Signaling Center, University of South Carolina , Medical University of South Carolina , and Clemson University , Charleston, South Carolina 29425, United States.
Biochemistry. 2014 Mar 4;53(8):1373-9. doi: 10.1021/bi401586h. Epub 2014 Feb 21.
Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ~10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca(2+) inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1-RyR2 chimeras in HEK293 cells and determined the Ca(2+) activation and inactivation affinities of these channels using the [(3)H]ryanodine binding assay. Replacing two distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity for Ca(2+) inactivation. The first region (RyR2 amino acids 4020-4250) contains two EF-hand Ca(2+) binding motifs (EF1, amino acids 4036-4047; EF2, amino acids 4071-4082), and the second region includes the putative second transmembrane segment (S2). A RyR1-backbone chimera containing only EF2 from RyR2 had a modest (not significant) change in Ca(2+) inactivation, whereas another chimera channel carrying only EF1 from RyR2 had a significantly reduced level of Ca(2+) inactivation. The results suggest that EF1 is a more critical determinant for RyR inactivation by Ca(2+). In addition, activities of the chimera carrying RyR2 EF-hands were suppressed at 10-100 μM Ca(2+), and the suppression was relieved by 1 mM Mg(2+). The same effects have been observed with wild-type RyR2. A mutant RyR1 carrying both regions replaced with RyR2 sequences (amino acids 4020-4250 and 4560-4618) showed a Ca(2+) inactivation affinity comparable to that of RyR2, indicating that these regions are sufficient to confer RyR2-type Ca(2+)-dependent inactivation on RyR1.
骨骼肌（RyR1）和心肌（RyR2）型兰尼碱受体钙通道对毫摩尔 Ca（2+）浓度敏感，但 RyR2 对抑制性 Ca（2+）的亲和力比 RyR1 低约 10 倍。先前的研究表明，兰尼碱受体的 C 端四分之一区域对于 Ca（2+）失活具有关键的结构域。为了进一步深入了解 Ca（2+）对 RyR 调节的分子基础，我们在 HEK293 细胞中构建和表达了 18 种 RyR1-RyR2 嵌合体，并使用 [(3)H]兰尼碱结合测定法确定了这些通道的 Ca（2+）激活和失活亲和力。用 RyR2 相应序列替换 RyR1 的两个不同区域会降低对 Ca（2+）失活的亲和力。第一个区域（RyR2 氨基酸 4020-4250）包含两个 EF 手 Ca（2+）结合基序（EF1，氨基酸 4036-4047；EF2，氨基酸 4071-4082），第二个区域包括假定的第二跨膜段（S2）。仅包含 RyR2 中 EF2 的 RyR1 骨架嵌合体在 Ca（2+）失活方面仅有适度（无统计学意义）的变化，而另一个携带 RyR2 中仅 EF1 的嵌合体通道的 Ca（2+）失活水平显著降低。结果表明，EF1 是 RyR 受 Ca（2+）失活的更关键决定因素。此外，携带 RyR2 EF 手的嵌合体通道的活性在 10-100 μM Ca（2+）下受到抑制，而 1 mM Mg（2+）可缓解这种抑制。野生型 RyR2 也观察到了相同的效果。携带 RyR2 序列（氨基酸 4020-4250 和 4560-4618）替换两个区域的突变型 RyR1 显示出与 RyR2 相当的 Ca（2+）失活亲和力，表明这些区域足以赋予 RyR1 型 RyR2 依赖的 Ca（2+）失活。
