CHLORIDE CHANNELS AND GASTRIC ACID SECRETION

这项建议的广泛和长期目标是确定 调节胃HC 1分泌的机制。 治疗控制 胃溃疡疾病依赖于控制HC 1 分泌物 而Mg ~（2+）依赖的、K ~+依赖的和H ~+转运的 胃H/K ATP酶（E.C. 1.2.1.36）负责生产HC 1 以ATP为代价，很少有人知道生理相关的 控制HC 1分泌的机制。 作为我们的主要工作假设， 壁细胞分泌膜Cl-通道活性 是调节哺乳动物中HCl分泌的主要靶标。 我们有 最近取得了一项突破，可以对这种情况进行严格的测试， 假设：胃囊泡Cl-通道的单通道记录 已经获得了与平面脂质双层融合的双层。 制备囊泡 从组胺处理的兔的胃粘膜。 这些囊泡 富含H/K ATP酶。 在刺激下，H/K ATP酶最大限度地 与分泌膜相连。 这些囊泡制剂是 因此富含分泌膜，并显示MgATP，K+和 Cl-依赖的HCl积累和K+和Cl-转运， 这是HCl分泌的刺激状态的特征。 根据我们 主要假设，这些囊泡应该含有受调节的Cl-通道。 关键的问题是，其中是否有任何一个代表了拟议的 刺激相关Cl-通道。 不管使用什么技术 测量单个Cl-通道，无论是膜片钳还是平面脂质双层， 有必要证明记录的通道来自于 细胞和膜的目的，并参与通道 感兴趣的分泌过程 幸运的是，兔子的壁细胞 提供了一个独特的有用的系统， 以及分泌功能的宏观测量的生物化学，如HC 1 记录细胞和囊泡水平的分泌和净KC 1通量 单一频道 具体目的是：1）在单个细胞中观察到Cl-通道 平面脂质双层中的通道记录将根据 到单沟道单元电导，电压依赖性，门控电荷， 对阴离子和阳离子的选择性，以及对通道阻滞剂的敏感性， 将比较这些单通道特性， 与2）Cl-通道宏观测量的特点对比 功能如细胞和囊泡的HCl积累和净KCl通量。 Cl-通道功能的网络行为和宏观测量将是 用于研究记录的单个Cl-通道在 调节HCl分泌。 3)刺激相关变化 Cl-通道行为将与刺激依赖性变化相关， Cl-通道功能的宏观测量（HCl积累和净KCl 通量），例如可能由电离的游离Ca 2+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Cl-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.