Bertl A, Slayman C L
Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1990 Oct;87(20):7824-8. doi: 10.1073/pnas.87.20.7824.
The vacuolar membrane of the yeast Saccharomyces cerevisiae, which is proposed as a system for functional expression of membrane proteins, was examined by patch-clamp techniques. Its most conspicuous feature, in the absence of energizing substrates, is a cation channel with a characteristic conductance of approximately 120 pS for symmetric 100 mM KCl solutions and with little selectivity between K+ and Na+ (PNa+/PK+ approximately 1) but strong selectivity for cations over anions (PCl-/PK+ less than 0.1). Channel gating is voltage-dependent; open probability, Po, reaches maximum (approximately 0.7) at a transmembrane voltage of -80 mV (cytoplasmic surface negative) and declines at both more negative and more positive voltages (i.e., to 0 around +80 mV). The time-averaged current-voltage curve shows strong rectification, with negative currents (positive charges flowing from vacuolar side to cytoplasmic side) much larger than positive currents. The open probability also depends strongly on cytoplasmic Ca2+ concentration but, for ordinary recording conditions, is high only at unphysiologically high (greater than or equal to 1 mM) Ca2+. However, reducing agents such as dithiothreitol and 2-mercaptoethanol poise the channels so that they can be activated by micromolar cytoplasmic Ca2+. The channels are blocked irreversibly by chloramine T, which is known to oxidize exposed methionine and cysteine residues specifically.
酿酒酵母的液泡膜被提议作为膜蛋白功能表达的一个系统，采用膜片钳技术对其进行了研究。在没有供能底物的情况下，其最显著的特征是一个阳离子通道，对于对称的100 mM KCl溶液，其特征电导约为120 pS，对K⁺和Na⁺之间的选择性很小（PNa⁺/PK⁺约为1），但对阳离子的选择性远高于阴离子（PCl⁻/PK⁺小于0.1）。通道门控是电压依赖性的；开放概率Po在跨膜电压为 -80 mV（细胞质表面为负）时达到最大值（约为0.7），在更负和更正的电压下均下降（即在 +80 mV左右降至0）。时间平均电流 - 电压曲线显示出强烈的整流作用，负电流（正电荷从液泡侧流向细胞质侧）远大于正电流。开放概率也强烈依赖于细胞质Ca²⁺浓度，但在普通记录条件下，仅在非生理性高浓度（大于或等于1 mM）的Ca²⁺时才较高。然而，诸如二硫苏糖醇和2 - 巯基乙醇等还原剂可使通道处于易于被微摩尔浓度的细胞质Ca²⁺激活的状态。这些通道被氯胺T不可逆地阻断，已知氯胺T能特异性氧化暴露的甲硫氨酸和半胱氨酸残基。
