MOLECULAR CLONING OF EPITHELIAL K CHANNELS

肾皮质集合管（CCT）中的钾通道在肾皮质的钾通道中起重要作用。 在肾脏钾（K）分泌中起重要作用， 保持身体的整体钾平衡。在这个肾单位片段中，钾 分泌速率取决于顶端膜K渗透性和细胞膜K-通道。 电化学驱动力K跨顶膜。的 与钾分泌相关的顶端膜钾渗透性似乎出现 从一个小的传导，温和向内整流通道， 正常情况下的高开概率（P-O）。然而， 该通道强烈依赖于细胞内pH。这在很大程度上 解释了临床观察结果， 大量钾流失和低钾血症 最近，两个密切相关的肾脏K通道已从大鼠中克隆， 命名为ROMK 1和ROMK 2。ROMK家族的生理相关性 与小电导顶端钾通道功能相似 负责哺乳动物CCT的K分泌和K循环， 塔尔预测的ROMK的一级序列和膜拓扑结构是 与可兴奋细胞的电压门控K通道完全不同。 然而，这两个ROMK克隆与R 0 MK克隆具有显著的同源性。 维持静息电位的内向整流（IRK）通道家族 在EK附近，并允许可兴奋细胞的长去极化反应。 本提案利用了结构和功能上的相似性， 在ROMK和IRK家庭之间解决一些重要问题， ROMK 2中的结构-功能关系。定点诱变将是 用于确定ROMK 2的区域和特定残基， 重要的是：（1）温和的内向整流，（2）强烈的pH依赖性， (3)渗透路径和选择性特征，（4）位点和亲和力 钡块。该提案还涉及结构性问题， 氨基和羧基末端中的特定残基与 通道的假定孔隙区域和已知的“向内整流器”位点 在第二跨膜区。K和之间的相互作用 传导通路内的钡也将作为一种方法进行研究 用于表征渗透路径的结构。该项目将 增强我们对钾稳态的基本过程的了解， 健康和疾病。

Potassium channels in the renal cortical collecting tubule (CCT) play an important role in potassium (K) secretion by the kidney and help to maintain the body's overall K balance. In this nephron segment, potassium secretion rates depend on both the apical membrane K permeability and the electrochemical driving forces for K across the apical membrane. The apical membrane K permeability relevant for K secretion appears to arise from a small conductance, mildly inward rectifying channel which has a high open probability (P-O) under normal conditions. However, the P-O of this channel is strongly dependent on intracellular pH. This largely explains the clinical observation that alkalosis is often associated with substantial K loss and hypokalemia. Recently two closely related renal K channels have been cloned from rat, designated ROMK1 and ROMK2. The physiological relevance of the ROMK family is its functional similarity to the small conductance apical K channels that are responsible for K secretion from mammalian CCT and K recycling in TALH. The predicted primary sequences and membrane topologies of ROMK are quite different from the voltage-gated K channels of excitable cells. However, both of these ROMK clones share significant homologies with the inward rectifier (IRK) family of channels that maintain resting potential near EK and permit long depolarizing responses in excitable cells. The present proposal exploits the structural and functional similarity between ROMK and IRK families to address some important issues regarding structure-function relations in ROMK2. Site directed mutagenesis will be used to determine the regions and specific residues of ROMK2 that are important for: (1) mild inward rectification, (2) strong pH dependence, (3) permeation path and selectivity characteristics, (4) site and affinity of barium block. The proposal also addresses structural issues involving the proximity of specific residues in the amino and carboxy termini to the putative pore region of the channel and to known "inward rectifier" sites in the second transmembrane spanning region. Interactions between K and barium within the conduction pathway will also be investigated as a method for characterizing the structure of the permeation path. The project will enhance our knowledge of basic processes underlying K homeostasis in health and disease.