Garty H, Yeger O, Asher C
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1988 Apr 25;263(12):5550-4.
Effects of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO) on the epithelial Na+ channel were evaluated by measuring the amiloride-blockable 22Na+ fluxes in membrane vesicles derived from the toad bladder epithelium. Incubating whole cells or isolated membranes with PGO readily and irreversibly blocked the channel-mediated tracer flux. Na+ ions present during the interaction of membranes with PGO could protect channels from inactivation by PGO. This effect required the presence of Na+ at the luminal side of the membrane and was characterized by an IC50 of 79 mM Na+. Amiloride, too, could desensitize channels to PGO, but its effect was significant only when whole cells were interacted with the protein-modifying reagent. The data are compatible with a model in which the conductive path of the channel contains a functional arginine, possibly forming a salt bridge with a carboxylic group, which is involved in Na+ translocation and amiloride binding. It was also shown that the augmentation of transport induced by incubating whole cells in Ca2+-free solution (Garty, H., and Asher, C. (1985) J. Biol. Chem. 260, 8330-8335) involves the activation or recruitment of channels that are not vulnerable to PGO prior to incubation.
通过测量源自蟾蜍膀胱上皮的膜囊泡中氨氯地平可阻断的(^{22}Na^+)通量，评估精氨酰和赖氨酰特异性试剂苯乙二醛（PGO）对上皮(Na^+)通道的影响。用PGO孵育全细胞或分离的膜会迅速且不可逆地阻断通道介导的示踪剂通量。膜与PGO相互作用期间存在的(Na^+)离子可保护通道免受PGO失活的影响。这种效应需要在膜的管腔侧存在(Na^+)，其特征是(Na^+)的半数抑制浓度（IC50）为79 mM。氨氯地平也可使通道对PGO脱敏，但其效应仅在全细胞与蛋白质修饰试剂相互作用时才显著。这些数据与一个模型相符，在该模型中，通道的传导路径包含一个功能性精氨酸，可能与一个羧基形成盐桥，该羧基参与(Na^+)转运和氨氯地平结合。研究还表明，在无钙溶液中孵育全细胞诱导的转运增强（Garty, H., and Asher, C. (1985) J. Biol. Chem. 260, 8330 - 8335）涉及在孵育前对PGO不敏感的通道的激活或募集。
