Sukhareva M, Morrissette J, Coronado R
Department of Physiology, University of Wisconsin School of Medicine, Madison 53706.
Biophys J. 1994 Aug;67(2):751-65. doi: 10.1016/S0006-3495(94)80536-3.
We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.
我们使用45Ca2+通量和单通道记录技术，研究了氯离子（Cl-）对兔骨骼肌连接肌质网（SR）钙（Ca2+）通透性的影响。在45Ca2+外流实验中，将含有5 mM 45Ca2+的150 mM单价盐溶液被动加载到SR内腔。在囊泡外0.4 - 0.8 microM游离Ca2+和加载到SR内腔的150 mM相同单价盐存在的情况下，通过快速过滤测量45Ca2+的释放。当单价盐在含有Cl-（Tris-Cl、胆碱-Cl、KCl）的SR中达到平衡，而不是有机阴离子或其他卤化物（葡萄糖酸盐-、甲磺酸盐-、乙酸盐-、HEPES-、Br-、I-）时，释放速率高出5 - 10倍。阳离子（K+、Tris+）可以互换，而对释放速率没有显著影响。为了确定Cl-是否刺激兰尼碱受体，我们测量了在不含Cl-和含Cl-的溶液中，ATP（总量5 mM）和咖啡因（总量20 mM）对释放的刺激作用以及Mg2+（估计游离量0.8 mM）的抑制作用。ATP、咖啡因和Mg2+的作用在K - 葡萄糖酸盐和Tris - 葡萄糖酸盐中最大，在KCl中居中，而在胆碱-Cl和Tris-Cl中明显较弱或不存在。普鲁卡因（10 mM）抑制了在K - 葡萄糖酸盐中测量的咖啡因刺激的释放，而Cl-通道阻滞剂氯贝酸（10 mM）而非普鲁卡因抑制了在胆碱-Cl中测量的对咖啡因不敏感的释放。钌红（20 microM）在所有溶液中均抑制释放。在与平面双层融合的SR中，我们鉴定出一种非选择性Cl-通道（PCl:PTris:PCa = 1:0.5:0.3），该通道被钌红和氯贝酸阻断，但不被普鲁卡因阻断。这些传导和药理学特性表明该通道可能介导Cl-依赖性SR Ca2+释放。Cl-对该通道开放概率无影响、普鲁卡因完全阻断以及氯贝酸刺激而非抑制，这些表明兰尼碱受体对Cl-依赖性释放无贡献。在非选择性通道和兰尼碱受体通道在终末池彼此独立运作的假设下，构建了Cl-依赖性释放的堵塞模型，即Cl-消除了大阴离子对非选择性通道的抑制作用。Cl-对SR Ca2+通透性的显著巨大贡献表明，非选择性Cl-通道可能在静息肌细胞中控制SR的Ca2+通透性。
