Edwards J C, Tulk B, Schlesinger P H
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Membr Biol. 1998 May 15;163(2):119-27. doi: 10.1007/s002329900376.
p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.
p64是一种通过从肾微粒体进行生化纯化而被鉴定为氯离子通道的蛋白质。我们使用重组痘苗病毒/T7 RNA聚合酶驱动系统在HeLa细胞中表达了p64。从感染/转染的细胞中制备总细胞膜，并将其融合到平面脂质双分子层中。在表达p64的细胞中发现了一种新的氯离子通道活性，而在对照细胞中未发现。与p64相关的活性表现出强烈的阴离子对阳离子选择性。单通道在正电位下具有42 pS的单通道电导，显示出明显的外向整流。氯离子通道活性通过用碱性磷酸酶处理膜而被激活，并被DNDS和TS-TM杯芳烃(4)抑制。通过氯离子外流测定法测定全膜阴离子通透性，结果显示表达p64的细胞的膜在氯离子通透性上有小幅但高度显著的增加，这与一种新的氯离子通道活性的表达一致。
