Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, USA.
Biophys J. 2012 Jul 18;103(2):202-11. doi: 10.1016/j.bpj.2012.06.001. Epub 2012 Jul 17.
Store-operated calcium entry (SOCE) is an important Ca(2+) entry pathway in skeletal muscle. However, direct electrophysiological recording and full characterization of the underlying SOCE current in skeletal muscle cells (I(SkCRAC)) has not been reported. Here, we characterized the biophysical properties, pharmacological profile, and molecular identity of I(SkCRAC) in skeletal myotubes, as well as the regulation of its rate of activation by temperature and the type I ryanodine receptor (RyR1). I(SkCRAC) exhibited many hallmarks of Ca(2+) release activated Ca(2+) currents (I(CRAC)): store dependence, strong inward rectification, positive reversal potential, limited cesium permeability, and sensitivity to SOCE channel blockers. I(SkCRAC) was reduced by siRNA knockdown of stromal interaction molecule 1 and expression of dominant negative Orai1. Average I(SkCRAC) current density at -80mV was 1.00 ± 0.05 pA/pF. In the presence of 20 mM intracellular EGTA, I(SkCRAC) activation occurred over tens of seconds during repetitive depolarization at 0.5Hz and was inhibited by treatment with 100 μM ryanodine. The rate of SOCE activation was reduced threefold in myotubes from RyR1-null mice and increased 4.6-fold at physiological temperatures (35-37°C). These results show that I(SkCRAC) exhibits similar biophysical, pharmacological, and molecular properties as I(CRAC) in nonexcitable cells and its rate of activation during repetitive depolarization is strongly regulated by temperature and RyR1 activity.
钙库操纵性钙内流（SOCE）是骨骼肌中一种重要的钙离子内流途径。然而，尚未有报道对骨骼肌细胞中的基础 SOCE 电流（I(SkCRAC)）进行直接电生理记录和全面特征描述。在此，我们对骨骼肌成肌细胞中 I(SkCRAC)的生物物理特性、药理学特性和分子特征进行了表征，以及其激活速率受温度和型 1 ryanodine 受体（RyR1）的调控。I(SkCRAC)表现出许多钙释放激活钙电流（I(CRAC)）的特征：依赖于钙库、强内向整流、正反转电位、有限的铯通透性以及对 SOCE 通道阻断剂的敏感性。I(SkCRAC)可通过基质相互作用分子 1 的 siRNA 敲低和显性负性 Orai1 的表达而减少。在-80mV 的平均 I(SkCRAC)电流密度为 1.00±0.05pA/pF。在 20mM 细胞内 EGTA 的存在下，I(SkCRAC)在 0.5Hz 的重复去极化期间数十秒内激活，并被 100μM ryanodine 处理所抑制。在 RyR1 缺失型小鼠的成肌细胞中，SOCE 激活速率降低了三倍，而在生理温度（35-37°C）下则增加了 4.6 倍。这些结果表明，I(SkCRAC)在非兴奋性细胞中表现出与 I(CRAC)相似的生物物理、药理学和分子特性，其在重复去极化期间的激活速率受温度和 RyR1 活性的强烈调控。
