Rebbeck Robyn T, Willemse Hermia, Groom Linda, Casarotto Marco G, Board Philip G, Beard Nicole A, Dirksen Robert T, Dulhunty Angela F
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN USA.
John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital, PO Box 334, Canberra, ACT 2601 Australia.
Skelet Muscle. 2015 Jul 22;5:23. doi: 10.1186/s13395-015-0049-3. eCollection 2015.
Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca(2+) release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1.
We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca(2+) currents and Ca(2+) transients in myotubes.
We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the β1a subunit. Activation of RyR2 by 10 nM β1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for β1a. A reduction in activation was also observed when 10 nM β1a was added to the alternatively spliced (ASI(-)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM β1a activation observed for both RyR2 and ASI(-)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca(2+) transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca(2+) release generated during skeletal EC coupling.
We conclude that the β1a subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1.
尽管骨骼肌中的兴奋-收缩（EC）偶联依赖于二氢吡啶受体（DHPR）对骨骼肌兰尼碱受体（RyR1）钙释放通道的物理激活，但DHPR与RyR1之间的激活途径仍不清楚。然而，该途径包括对EC偶联不可或缺并激活RyR1的DHPRβ1a亚基。在本论文中，我们探究了β1a激活RyRs的亚型特异性以及RyR1上的β1a结合位点。
我们使用脂质双层来测量单通道电流，并使用全细胞膜片钳来测量肌管中的L型钙电流和钙瞬变。
我们证明，磷脂双层中的骨骼肌RyR1和心脏RyR2通道均被10 - 100 nM的β1a亚基激活。10 nMβ1a对RyR2的激活作用小于对RyR1的激活作用，表明RyR2对β1a的亲和力降低。当将10 nMβ1a添加到缺乏ASI残基（A3481 - Q3485）的RyR1可变剪接（ASI(-)）亚型中时，也观察到激活作用降低。值得注意的是，RyR2的等效区域也缺少五个ASI残基中的四个，这表明这些残基的缺失可能导致与ASI(+)RyR1相比，RyR2和ASI(-)RyR1在10 nMβ1a激活时出现降低的情况。我们还研究了RyR1的一个多碱性基序（PBM）（K3495KKRRDGR3502）的影响，该基序位于ASI残基的紧邻下游，并与EC偶联有关。我们证实，中和PBM中的碱性残基（RyR1 K - Q）会导致在RyR1基因缺失（dyspedic）肌管中表达后钙瞬变幅度降低约50%，并且PBM对于脂质双层中RyR1通道的β1a亚基激活也是必需的。这些结果表明，去除β1a亚基与RyR1中PBM的相互作用可能直接导致骨骼肌EC偶联过程中约50%的钙释放。
我们得出结论，β1a亚基可能与RyR1和RyR2中基本保守的区域结合，并且该区域受RyR1中ASI残基和PBM的存在影响。
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