CHLORIDE CHANNELS AND GASTRIC ACID SECRETION

这项提案的广泛、长期目标是定义 胃酸分泌的调节机制。治疗控制 胃溃疡疾病依赖于控制HCl的干预措施 分泌物。而依赖镁的、依赖钾的和氢的转运 胃H/K-ATPase(E.C.1.2.1.36)负责HCl的产生 以ATP为代价，人们对生理上的相关知识知之甚少 控制HCl分泌的机制。作为我们的主要工作假设， 壁细胞分泌膜上氯离子通道的活性 是哺乳动物体内HCl分泌调节的主要靶点。我们有 最近实现了一项突破，使这一点得到了严格的考验 假设：胃小泡氯离子通道的单通道记录 得到了融合到平面类脂双分子膜上。制备了囊泡 从组胺处理的兔的胃粘膜中提取。这些小泡是 富含H/K-ATPase。在刺激下，H/K-ATPase最大限度地 与分泌膜相关的。这些泡腾剂是 因此富含在分泌膜中，并表现出镁三磷酸腺苷、钾和 依赖于氯的HCl积累和K和Cl-的运输 具有HCl分泌刺激状态的特点。根据我们的说法 主要假设是，这些小泡应该含有受调控的氯离子通道。 关键问题是，其中是否有任何一项代表了拟议的 刺激相关的氯离子通道。不管使用哪种技术 测量单个氯离子通道，无论是膜片钳还是平面脂质双层， 有必要证明所记录的频道来自 感兴趣的细胞和膜，并且通道参与 感兴趣的秘密过程。幸运的是，兔子的壁细胞 提供了一个独特有用的系统来将生理学、药理学、 和宏观分泌功能指标的生物化学，如HCl 记录细胞和囊泡水平的分泌物和Kc1净通量 单通道。具体目标是：1)在单细胞中观察到的氯通道 平面脂质双层中的通道记录的特征如下 对于单通道单位电导、电压依赖性、选通电荷， 对阴阳离子的选择性以及对通道阻滞剂和 PH值的变化。将对这些单通道特性进行比较并 对比2)氯离子通道宏观测量的特点 功能，如HCl积累和细胞和囊泡的Kc1净通量。 氯离子通道功能的净行为和宏观措施 用来研究记录的单个氯离子通道在 调节HCl的分泌。3)与刺激相关的变化 CL通道行为将与刺激依赖的变化相关 氯离子通道功能的宏观测量(HCl累积和净Kc1 助熔剂)，例如可能由电离的游离钙提供，或 蛋白激酶C、蛋白激酶A或G蛋白。初步结果： 抑制剂和pH敏感性表明记录的单个氯离子通道 可能参与了HCl的积累。

The broad, long term objectives of this proposal are to define the mechanisms of regulation of gastric HC1 secretion. Therapeutic control of gastric ulcer disease relies upon interventions which control HC1 secretion. Whereas the Mg2+-dependent, K+ dependent, and H+ transporting gastric H/K ATPase (E.C. 1.2.1.36) is responsible for the production of HC1 at the expense of ATP, little is known of the physiologically relevant mechanisms of control of HC1 secretion. As our major working hypothesis, the activity of Cl- channels of the secretory membrane of the parietal cell is a major target of regulation of HC1 secretion in mammals. We have recently achieved a breakthrough which allows a rigorous test of this hypothesis: Single channel recordings of Cl- channels of gastric vesicles fused to planar lipid bilayers have been obtained. Vesicles were prepared from the gastric mucosa of histamine treated rabbits. These vesicles are enriched in H/K ATPase. Upon stimulation, the H/K ATPase is maximally associated with the secretory membrane. These vesicle preparations are therefore enriched in secretory membranes, and exhibit MgATP, K+ and Cl-dependent HC1 accumulation and K+ and Cl- transport which are characteristic of the stimulated state of HC1 secretion. According to our major hypothesis, these vesicles should contain regulated Cl- channels. The key question is whether any of these represent the proposed stimulus-associated Cl- channel. Regardless of the technique used to measure single Cl- channels, whether patch clamp or planar lipid bilayers, it is necessary to demonstrate that the recorded channels arise from the cell and membrane of interest, and that the channel participates in the secretory process of interest. Fortunately, the rabbit parietal cell provides a uniquely useful system to relate the physiology, pharmacology, and biochemistry of macroscopic measures of secretory function such as HC1 secretion and net KC1 flux at the level of the cell and vesicle to recorded single channels. The Specific Aims are 1) Cl- channels observed in single channel recordings in planar lipid bilayers will be characterized according to single channel unit conductance, voltage dependence, gating charge, selectivity for anions and cations, and sensitivity to channel blockers and changes in pH. These single channel characteristics will be compared and contrasted with 2) characteristics of macroscopic measures of Cl- channel function such as HC1 accumulation and net KC1 flux by cells and vesicles. Net behavior and macroscopic measures of Cl- channel function will be employed to investigate the role of recorded single Cl- channels in regulated HC1 secretion. 3) Stimulus associated changes in single Cl-channel behavior will be correlated with stimulus dependent changes in macroscopic measures of Cl- channel function (HC1 accumulation and net KC1 flux) such as might be afforded by ionized free Ca2+, or effectors of protein kinase C, protein kinase A, or G-proteins. Preliminary results of inhibitor and pH sensitivity suggest that the recorded single Cl- channels may participate in HC1 accumulation.