MOLECULAR CLONING OF EPITHELIAL K CHANNELS

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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.