Xu Le, Gomez Angela C, Pasek Daniel A, Meissner Gerhard, Yamaguchi Naohiro
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, United States.
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, United States; Cardiac Signaling Center, University of South Carolina, Medical University of South Carolina and Clemson University, Charleston, SC 29425, United States.
Cell Calcium. 2017 Sep;66:62-70. doi: 10.1016/j.ceca.2017.05.013. Epub 2017 Jun 6.
The mammalian ryanodine receptor Ca release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2μM Ca, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1-3725 and RyR2 C-terminal aa 3692-4968 were inhibited by CaM at <1μM Ca similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1-4301 and RyR2 4254-4968 was activated at <1μM Ca similar to RyR1. Replacement of RyR1 aa 3726-4298 with corresponding residues from RyR2 conferred CaM inhibition at <1μM Ca, which suggests RyR1 aa 3726-4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca binding motifs in RyR1 aa 4081-4092 (EF1) and aa 4116-4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca concentrations.
哺乳动物的雷诺丁受体钙释放通道（RyR）有一个单一的保守高亲和力钙调蛋白（CaM）结合结构域。然而，在亚微摩尔钙浓度下，骨骼肌RyR1被CaM激活，而心肌RyR2被CaM抑制。这表明亚型特异性结构域参与了CaM对RyR的调节。为了深入了解CaM对心肌和骨骼肌RyR的差异调节，将RyR1/RyR2嵌合体和突变体在HEK293细胞中表达，并使用脂质双层方法测量它们的单通道活性。所有RyR1/RyR2嵌合体和突变体在2μM钙时都被CaM抑制，这与在微摩尔钙浓度下CaM对RyR1和RyR2的抑制作用一致。一个具有RyR1 N端氨基酸残基（aa）1-3725和RyR2 C端aa 3692-4968的RyR1/RyR2嵌合体在<1μM钙时被CaM抑制，类似于RyR2。相反，一个具有RyR1 aa 1-4301和RyR2 4254-4968的RyR1/RyR2嵌合体在<1μM钙时被激活，类似于RyR1。用RyR2的相应残基替换RyR1 aa 3726-4298可在<1μM钙时赋予CaM抑制作用，这表明RyR1 aa 3726-4298是CaM激活所必需的。对RyR1 aa 4081-4092（EF1）和aa 4116-4127（EF2）中两个预测的钙结合基序中的其他RyR1/RyR2嵌合体和突变体的表征表明，在亚微摩尔钙浓度下，EF手基序和大N端区域中的其他序列对于CaM对RyR1和RyR2的亚型特异性调节都是必需的。
