VOLTAGE-GATED PROTON CHANNELS IN ALVEOLAR EPITHELIUM

电压激活的质子选择电流最早于1982年在蜗牛神经元中被发现，在包括大鼠肺泡上皮(DeCoursey，1991)和人中性粒细胞(DeCoursey and Cherny，1993)在内的几种哺乳动物细胞的膜片钳研究中已经发现。这些H+通道是通过膜去极化、细胞质酸化和细胞外碱化的组合来开放的。它们似乎被设计成从细胞中排出酸。在吞噬细胞中，它们在呼吸爆发时被激活，这是杀死细菌的过程，因此它们促进了炎症反应。一般而言，质子通道在高代谢活动期维持细胞内的动态平衡。与大多数离子通道相比，人们对这些通道的了解更少。H+通道分子还没有得到明确的鉴定，部分原因是没有有效和特异的抑制剂。本项目的目的之一是研究有机药物，如局麻药对H+通道的抑制作用。有机化合物(如局麻药或金刚烷胺)与通道分子相互作用的机制将揭示质子通过膜的传导途径的性质。最有效和最知名的H+通道抑制剂之一是锌离子。我们将探索锌离子效应强烈pH依赖性的机制，并测试锌离子与通道内外不同部位结合的假设，可能是控制门控电压依赖性的精致pH敏感性的相同调控部位(Cherny等人，1995)。我们将使用修饰特定氨基酸(组氨酸和半胱氨酸)的试剂来确定这些氨基酸是暴露在外部还是内部溶液中，以及它们在门控或渗透中扮演什么角色。将使用几种类型的噪声分析以及直接测量来探索单个H+通道(以前未被检测到)的行为。证明H+电流波动是由H+通道的随机选通引起的，这将提供强有力的证据，证明该分子是离子通道而不是载体。

First described in snail neurons in 1982, voltage-activated proton-selective currents have been found in patch-clamp studies of several mammalian cells, including rat alveolar epithelium (DeCoursey, 1991) and human neutrophils (DeCoursey and Cherny, 1993). These H+ channels are opened by a combination of membrane depolarization, cytoplasmic acidification, and extracellular alkalinization. They appear to have been designed to extrude acid from cells. In phagocytes they are activated during the respiratory burst, the process of killing bacteria, thus they facilitate the inflammatory response. In general, proton channels serve to maintain homeostasis in cells during periods of high metabolic activity. Less is known about these channels than about most ion channels. The H+ channel molecule has not been positively identified, partly because there are no potent and specific inhibitors. One aim of this project is to characterize the inhibition of H+ channels by organic drugs, such as local anesthetics. The mechanism of interaction between organic compounds (e.g., local anesthetics or amantidine) and the channel molecule will reveal clues to the nature of the conduction pathway by which protons cross the membrane. One of the most potent and best-known inhibitors of H+ channels is Zn2+. We will explore the mechanism of the strong pH dependence of Zn2+ effects, and test the hypothesis that Zn2+ binds to distinct sites at the external and internal side of the channel, perhaps the same regulatory sites that control the exquisite pH sensitivity of the voltage dependence of gating (Cherny et al, 1995). We will use reagents that modify specific amino acids (histidine and cysteine) to determine whether these amino acids are exposed to the external or internal solution, and what roles they play in gating or permeation. The behavior of single H+ channels (which have not been detected previously) will be explored using several types of noise analysis as well as by direct measurement. Demonstration that H+ current fluctuations result from stochastic gating of H+ channels will provide strong evidence that the molecule is an ion channel rather than a carrier.