Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.
Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.
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DOI：
10.1016/s0006-3495(94)80536-3
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发表时间：
1994-08
影响因子：
3.4
通讯作者：
M. Sukhareva;J. Morrissette;R. Coronado
中科院分区：
文献类型：
--
作者：
M. Sukhareva;J. Morrissette;R. Coronado
采用~（45）Ca ~（2+）通量和单通道记录技术，研究了Cl ~-对家兔骨骼肌连接肌浆网（SR）Ca ~（2+）通透性的影响。在45 Ca 2+流出实验中，SR的内腔被动地装载有含有5 mM 45 Ca 2+的150 mM单价盐的溶液。在囊外0.4-0.8 μ M游离Ca 2+和150 mM加载到SR腔中的相同单价盐存在下，通过快速过滤测量45 Ca 2+的释放。当一价盐平衡于含SR的Cl-（Tris-Cl、胆碱-Cl、KCl）而不是有机阴离子或其它卤化物（葡糖酸盐-、甲磺酸盐-、乙酸盐-、HEPES-、Br-、I-）时，释放速率高5-10倍。阳离子（K+、Tris+）可以互换，对释放速率没有显着影响。为了确定Cl-是否刺激ryanodine受体，我们测量了在无Cl（-）和含Cl（-）溶液中ATP（5 mM总量）和咖啡因（20 mM总量）对释放的刺激以及Mg 2+（0.8 mM估计游离）对释放的抑制。ATP、咖啡因和Mg ~（2+）对葡萄糖酸钾和葡萄糖酸三羟甲基氨基甲烷的影响最大，对氯化钾的影响居中，对胆碱-Cl和Tris-Cl的影响最小或不存在。普鲁卡因（10 mM）抑制咖啡因刺激的释放测量葡萄糖酸钾，而氯通道阻滞剂氯贝酸（10 mM），但不普鲁卡因抑制咖啡因不敏感的释放测量胆碱-Cl。钌红（20 μ M）抑制释放在所有的解决方案。在SR融合到平面双层，我们确定了一个非选择性的氯离子通道（PCl：PTris：PCa=1：0.5：0.3）阻断钌红和氯贝酸，但不是普鲁卡因。这些导电和药理学性质表明该通道可能介导Cl（-）依赖性SR Ca ~（2+）释放。氯离子对该通道开放概率无影响，普鲁卡因完全阻断，氯贝酸刺激而非抑制，表明兰尼碱受体对氯离子依赖性释放无贡献。一个插头模型的Cl（-）-依赖性的释放，其中Cl-消除了抑制的非选择性通道的大阴离子，制定的假设下，非选择性通道和兰尼碱受体通道相互独立的终端池。Cl-对SR Ca ~（2+）通透性的显著贡献表明，非选择性Cl-通道可能控制静息肌细胞SR的Ca ~（2+）通透性。
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.