Hamilton S L, Alvarez R M, Fill M, Hawkes M J, Brush K L, Schilling W P, Stefani E
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Anal Biochem. 1989 Nov 15;183(1):31-41. doi: 10.1016/0003-2697(89)90167-x.
Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [3H]PN200-110, and the alkaloid, [3H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling.
对源自管状（T）网络或肌浆网（SR）的骨骼肌膜进行了如下特性研究：二氢吡啶[3H]PN200 - 110和生物碱[3H]ryanodine的结合、多肽组成以及离子通道活性。讨论了优化这些放射性配体结合的条件。描述了一种双层脉冲技术，并用于检测这些膜中存在的通道。T小管膜融合到双层膜中显示存在氯离子通道和具有三种不同电导的二氢吡啶敏感钙通道。对二氢吡啶敏感通道的电压依赖性进行了进一步表征。脉冲实验表明存在两种不同群体的二氢吡啶敏感通道。重肌浆网囊泡的融合显示出三种不同的离子通道；推测的钙释放通道、钾通道和氯离子通道。因此，这种分级分离程序提供了T小管和肌浆网膜，结合放射性配体结合和单通道记录技术，为研究骨骼肌离子通道的特性及其在兴奋 - 收缩偶联中的可能作用提供了一种有用的工具。
