Smith J S, Coronado R, Meissner G
Nature. 1985;316(6027):446-9. doi: 10.1038/316446a0.
Rapid calcium efflux from the sarcoplasmic reticulum (SR) is a necessary step in excitation-contraction coupling in skeletal muscle and is thought to be mediated by a calcium channel. Calcium efflux has been studied in fragmented SR vesicles by radioisotope efflux and fluorescence measurements. Several laboratories have reported that adenine nucleotides can stimulate calcium efflux from SR. In recent reports, Ca2+ release with a first-order rate constant as high as 100 s-1 has been observed for nucleotide-stimulated Ca2+ release from SR vesicles. Also, radioisotope efflux was blocked by Mg2+ and micromolar concentrations of the polycationic dye, ruthenium red. These high rates of transport are difficult to reconcile with a mechanism other than passive diffusion through a nucleotide-activated 'calcium release channel'. Using the fusion technique for inserting SR proteins into planar lipid bilayers, we report here single-channel recordings of calcium release channels from purified 'heavy' SR membranes. Channels have been identified on the basis of their activation by adenine nucleotides, blockade by ruthenium red, and selectivity for divalent cations. Surprisingly, the channel studied here exhibits an unusually large conductance of 170 pS in 50 mM Ba2+ while still being capable of discriminating against monovalent cations by a permeability ratio, P(Ba)/P(Cs) = 11.4.
肌浆网(SR)中钙离子的快速外流是骨骼肌兴奋-收缩偶联的必要步骤,并且被认为是由一种钙通道介导的。已通过放射性同位素外流和荧光测量在破碎的SR囊泡中研究了钙离子外流。几个实验室报告称,腺嘌呤核苷酸可以刺激SR中的钙离子外流。在最近的报告中,已观察到从SR囊泡中核苷酸刺激的Ca2+释放具有高达100 s-1的一级速率常数的Ca2+释放。此外,放射性同位素外流被Mg2+和微摩尔浓度的聚阳离子染料钌红阻断。这些高转运速率很难与通过核苷酸激活的“钙释放通道”进行被动扩散以外的机制相协调。使用将SR蛋白插入平面脂质双层的融合技术,我们在此报告了从纯化的“重”SR膜中记录的钙释放通道的单通道记录。通道已根据它们被腺嘌呤核苷酸激活、被钌红阻断以及对二价阳离子的选择性来鉴定。令人惊讶的是,此处研究的通道在50 mM Ba2+中表现出异常大的170 pS电导,同时仍能够通过渗透比P(Ba)/P(Cs)=11.4区分单价阳离子。