VOLTAGE ACTIVATED PROTON CHANNELS IN ALVEOLAR EPITHELIUM

电压激活的H+电流将在大鼠肺泡上皮细胞中进行研究 原代培养中的细胞，使用全细胞和切除的贴片 gigohm-seal技术的配置。肺泡上皮细胞 产生并分泌肺表面活性物质， 在呼气时崩溃。肺泡上皮也防止水肿 通过吸收液体，并调节pH值和电解质组成， 肺泡亚相。最近在哺乳动物细胞中描述的H+电流， 用于从细胞中挤出酸。本项目将探讨 H+传导机制（gH）与H+的协同调节 通道门控由pH-0、PH-1和电压。 电压依赖性是否已设置 通过“质子阱”机制，其中膜电压和H+ 浓度是否具有动力学等效效应？有没有分开的 通道内外侧的变构质子化位点？ 这些结果将有助于确定gH的条件， 在完整的细胞中激活。 目前尚不清楚H+电流是否 由离子通道介导，或质子如何渗透假定的通道。 一个基本假设是电压激活的H+通道 不是简单的充满水的气孔，而是由“质子线”操作的。 一种质子跳过氢键链的机制， 至少部分的整合膜蛋白内的侧基。这 一个机制不同于“水线”机制，质子从一个跳跃 水分子到下一个，通过它H+渗透其他种类的离子 这些通道是充满水的孔隙。单通道电流将是 如果可能的话，直接记录，利用最近的改进， 膜片钳电路和石英移液器。 单个H+的行为 还将使用稳态H+电流噪声（方差）研究通道 在不同的温度、pH-0和pH-1下以及在 氘，来定义质子传导的性质。的行为 将测定生理温度下的H+通道。GH， 其电压依赖性由pH-O和pH-i设定， 生物传感器研究肺泡上皮细胞的反向转运。基本 这种重要的pH调节转运蛋白的特性，包括其 依赖于内部和外部[H+]和[Na+]，以及其可能的 通过磷酸化的调节将使用这种新的方法来检查。 Na+-H+反向转运具有速率决定电压的可能性- 将测试其反应循环中的依赖步骤。

Voltage-activated H+ currents will be studied in rat alveolar epithelial cells in primary culture, using the whole-cell and excised patch configurations of the gigohm-seal technique. Alveolar epithelial cells produce and secrete pulmonary surfactant, which keeps alveoli from collapsing during expiration. The alveolar epithelium also prevents edema by absorbing fluid, and regulates the pH and electrolyte composition of the alveolar subphase. H+ currents, described recently in mammalian cells, serve to extrude acid from cells. This project will explore the nature of the H+ conductance mechanism (gH) and the cooperative regulation of H+ channels gating by pH-O, PH-i, and voltage. Is the voltage-dependence set by a "proton well" mechanism, in which membrane voltage and H+ concentration have kinetically equivalent effects? Are there separate allosteric protonation sites on the inner and outer sides of the channel? The results will help define the conditions under which the gH is activated in intact cells. It is not known whether H+ currents are mediated by ion channels, or how protons permeate the putative channels. A fundamental hypothesis proposed is that voltage-activated H+ channels are NOT simple water-filled pores, but operate by a "proton-wire" mechanism in which protons jump across a hydrogen-bonded chain formed at least partially of side groups within an integral membrane protein. This mechanism differs from a "water-wire" mechanism, protons hopping from one water molecule to the next, by which H+ permeates other kinds of ion channels which ARE water-filled pores. Single-channel currents will be recorded directly if possible, taking advantage of recent improvements in patch-clamp circuitry and quartz pipettes. The behavior of single H+ channels will also be studied using stationary H+ current noise (variance) analysis at varying temperatures, pH-O and PH-i and in the presence of deuterium, to define the nature of the proton conductance. The behavior of H+ channels at physiological temperatures will be determined. The gH, whose voltage dependence is set by pH-O and pH-i will be used as a biological sensor to study antiport in alveolar epithelium. Fundamental properties of this important pH-regulating transporter, including its dependence on internal and external [H+] and [Na+], and its possible regulation by phosphorylation will be examined using this novel approach. The possibility that Na+-H+ antiport has a rate-determining voltage- dependent step in its reaction cycle will be tested.