Department of Translational Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia.
FASEB J. 2012 Dec;26(12):5049-59. doi: 10.1096/fj.12-211334. Epub 2012 Sep 7.
Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the β(1a) subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide β(1a)V490-M524, corresponding to the C-terminal 35 residues of β(1a), reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in β(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type β(1a)A474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue β(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.
骨骼肌的兴奋-收缩（EC）偶联依赖于横管二氢吡啶受体（DHPR）电压传感器和细胞内兰尼碱受体（RyR1）钙释放通道之间的蛋白相互作用。我们提出了新的数据，表明β（1a）亚基的 C 端 35 个残基采用新生的α-螺旋，其中 3 个疏水性残基排列形成疏水面，与兔骨骼肌分离的 RyR1 结合。肽β（1a）V490-M524 中 C 端 35 个残基的疏水性残基（L496、L500、W503）的突变（对应于β（1a）的 C 端 35 个残基），将肽与 RyR1 的结合减少到 15.2±7.1%，并阻止了 10 nM 野生型肽对 RyR1 的 2.9±0.2 倍激活。在β（1a）与 RyR1 结合中涉及的上游疏水性七肽重复序列对 RyR1 激活没有贡献。含有七肽重复序列和疏水面残基的野生型β（1a）A474-A508 肽（10 nM），在突变七肽重复序列残基后，RyR1 活性分别增加了 2.3±0.2-和 2.2±0.3 倍。我们得出结论，β（1a）C 端 35 个残基中的特定疏水面残基与 RyR1 结合，并增加脂质双层中的通道活性，因此可能支持骨骼肌 EC 偶联。
