MOLECULAR CLONING OF EPITHELIAL K CHANNELS

肾皮质集合管(CCT)中的钾通道发挥作用 肾脏在钾(K)分泌中的重要作用并有助于 保持身体的整体钾平衡。在这段肾单位中，钾 分泌速率取决于根尖膜钾的通透性和 钾在根尖膜上的电化学驱动力。这个 与钾分泌有关的根尖膜钾通透性似乎出现上升 从一个小电导、温和的内向整流通道 正常情况下高开路概率(P-O)。然而，中国的P-O 该通道强烈依赖于细胞内的pH。这在很大程度上 解释了碱中毒的临床观察 大量钾丢失和低钾血症。 最近从大鼠中克隆了两个密切相关的肾脏钾通道， 指定ROMK1和ROMK2。ROMK家族的生理学相关性 它的功能与小电导心尖K通道相似吗 它们负责哺乳动物CCT的K分泌和K的循环 说吧。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通道有很大不同。 然而，这两个ROMK克隆与 保持静息电位的内向整流(IRK)通道系列 在EK附近，并允许在可兴奋细胞中进行长时间的去极化反应。 目前的建议利用了结构和功能的相似性 ROMK和IRK家族之间的合作，以解决以下一些重要问题 ROMK2中的结构-功能关系。定点突变将是 用于确定ROMK2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)位置和亲和力 这是一块钡块。该提案还涉及结构性问题，包括 氨基和羧基末端的特定残基与 通道的推定孔区和已知的“内向整流”位置 在第二跨膜区域。K和K之间的相互作用 传导途径中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 增强我们对钾动态平衡的基本过程的了解 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.