CHLORIDE CHANNELS AND GASTRIC ACID SECRETION

胃溃疡疾病的治疗控制依赖于控制HCl分泌的干预措施。这项提案的广泛、长期目标是定义参与调节HCl分泌的分子和机制。HCl的分泌涉及胃H/K ATPase和K的运输系统，K供应H/K ATPase，而C 1为HCl的产生提供等量的C 1。这一建议中要检验的工作假设是，受刺激的兔胃壁细胞的顶膜含有与调节胃HCl分泌密切相关的C1和K通道，并对其至关重要。C1通道C1C-2G是本实验室克隆的一种受钙离子或镁离子、CaMKII以及PKA、电压和胞外pH控制的整流子。钾通道可能是一种受PKA和细胞外pH调节的内向整流性钾通道。二价阳离子也可能起到一定作用。获得了一个候选K通道克隆RBHIK1，并将其特性与天然通道的特性进行了比较。生理上相关的K和C1向外运动基本上由相同的效应器调节，并对电气和化学环境的变化做出类似的反应。计划中的研究旨在了解促进整流的细胞内离子之间的相互作用，蛋白激酶、电压和pH在CIC-2G促进C1外向运动和K通过天然和克隆的内向整流钾通道中的作用。功能研究将得到使用包含阳离子结合位点、磷酸化位点和pH传感器位点突变的通道cDNA的相关结构研究的补充。对于CI和K通道，其具体目的是研究：1)阳离子在通道整流中的作用；2)蛋白激酶在C_1和K通道功能中的作用；3)质子在C_1和K通道功能中的作用；以及4)通道在胃壁细胞中处于非分泌(静息)和分泌(刺激)状态的位置。前三个目标将使用功能和相关的结构方法进行研究。目标4将使用基于通道序列知识的结构探针来实现。这些研究致力于解决与调节HCl分泌有关的分子和机制的基本问题。

Therapeutic control of gastric ulcer disease depends upon interventions which control HCl secretion. The broad, long term objectives of this proposal are to define the molecules and mechanisms involved in regulated HCl secretion. HCl secretion involves the gastric H/K ATPase and a transport system for K to supply the H/K ATPase and for C1 to provide equivalents of C1 for HCl production. The working hypothesis to be tested in this proposal is that the apical membrane of the stimulated rabbit gastric parietal cell contains both C1 and K channels which are intimately associated with and essential for regulated gastric HCl secretion. The C1 channel, C1C-2G was cloned in this laboratory, and it is a rectifier which is controlled by Ca2+ or Mg2+, CaMKII as well as PKA, voltage, and extracellular pH. The K channel appears to be an inwardly rectifying K channel which is regulated by PKA and extracellular pH. Divalent cations may also play a role. A candidate K channel clone, RBHIK1, has been obtained and its properties will be compared and contrasted with those of the native channel. The physiologically relevant outward movement of both K and C1 are regulated by essentially the same effectors and respond similarly to changes in the electrical and chemical environments. The planned studies are directed toward understanding the interactions between intracellular ions in promoting rectification, the effects of protein kinases, voltage, and pH in promotion of outward movements of C1 by CIC-2G and of K by the native and cloned inwardly rectifying K channel. Functional studies will be complemented by correlated structural studies using channel cDNAs containing mutations of the cation binding sites, phosphorylation sites, and pH sensor sites. The Specific Aims which apply to both the CI and K channels are to study: 1) the role of cations in rectification of the channels; 2) the role of protein kinases in C1 and K channel function; 3) the role of protons in C1 and K channel function; and 4) the location of the channels in the gastric parietal cell in the non-secreting (resting) and secreting (stimulated) states. The first 3 aims will be studied using functional as well as correlated structural approaches. Aim 4 will be accomplished using structural probes based on knowledge of the sequence of the channels. These studies strive to address the fundamental questions regarding the molecules and mechanisms involved in regulated HCl secretion.