VOLTAGE ACTIVATED PROTON CHANNELS IN ALVEOLAR EPITHELIUM

大鼠肺泡上皮细胞电压激活的H+电流的研究 原代培养的细胞，使用全细胞贴片和切除贴片 吉欧姆密封技术的配置。肺泡上皮细胞 产生和分泌肺表面活性物质，防止肺泡 在保质期内坍塌。肺泡上皮也可以防止浮肿。 通过吸收液体，并调节pH值和电解液组成 肺泡亚时相。最近在哺乳动物细胞中描述的H+电流， 用于从细胞中排出酸。这个项目将探索 H+电导机制(Gh)与H+的协同调节 通过pH-O、PH-I和电压进行通道选通。是电压依赖集吗？ 通过“质子井”机制，其中膜电压和H+ 浓度在运动学上有相同的影响吗？有单独的吗？ 通道内外两侧的变构质子化位置？ 这些结果将有助于定义Gh是在什么条件下 在完整的细胞中被激活。目前尚不清楚H+电流是否 由离子通道或质子如何渗透到假定的通道。 提出的一个基本假设是电压激活的H+通道 不是简单的充满水的毛孔，而是由一根“质子线”操作的。 一种质子跳过氢键链的机制 在一个完整的膜蛋白中至少有部分侧基。这 机制不同于“水线”机制，质子跳跃不同于 水分子，H+通过它渗透到其他类型的离子 孔道是充满水的毛孔。单通道电流将是 如果可能，直接记录，利用最近在 膜片钳电路和石英移液管。单H~+的行为 还将使用平稳的H+电流噪声(方差)来研究通道 在不同温度、pH-O和PH-I以及在 氢，用来定义质子电导的性质。的行为 将测定生理温度下的H+通道。The Gh， 其电压依赖关系由pH-O和pH-I设置，将用作 生物传感器用于研究肺泡上皮细胞的拮抗作用。基本原理 这种重要的pH调节转运蛋白的性质，包括它的 对内外[H+]和[Na+]的依赖及其可能 将使用这种新的方法来研究磷酸化的调节。 Na+-H+反端口具有决定速率的电压的可能性- 将对其反应周期中的相关步骤进行测试。

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.