Renal ATP- sensitive, inwardly rectifying K+ (KATP) channels not only provide the final pathway for regulated K+ secretion by principal cells, but also play a vital role in thick ascending limb NaCl absorption, which is crucial to overall salt balance, as well as urinary dilution and concentration. Renal secretory KATP channels are regulated by phosphorylation-dephosphorylation processes resulting from receptor- mediated activation of protein kinase A (PKA) and C (PKC), by non- hydrolytic nucleotide (MgATP) interactions which link the channel to metabolic processes within these epithelial cells, and by alterations in cytosolic pH which could contribute to abnormal K+ excretion in acid-base disturbances. The inwardly rectifying KATP channel, ROMK, cloned from rat kidney exhibits functional-regulatory properties virtually identical to the low conductance secretory KATP channel in principal and TAL cells, and provides the basis for studying the specific mechanisms involved in these regulatory processes at a molecular level. The ROMK gene contains a number of exons that give rise to at least four distinct alternatively spliced transcripts that are differentially expressed along the nephron, and that encode channel proteins differing in their amino- termini. A major hypothesis is that this molecular diversity provides for channel functional-regulatory diversity. Electrophysiological (patch and whole cell voltage clamping) and molecular/biochemical (site- directed mutagenesis, chimeras, phosphopeptide mapping) techniques adapted to Xenopus oocytes and HEK cells transiently expressing wild- type or epitope-tagged ROMK constructs will be used to: i) study isoform-specific regulation by kinases, and non-hydrolytic ATP and H+ interactions; ii) determine the channel domains and specific amino acids involved in phosphorylation by these kinases, in binding MgATP and in titration of H+; iii) assess the functional-regulatory consequences of interactions (heteromeric complexes) between/among channel isoforms; and iv) clone the moderate conductance 70 pS KATP expressed on apical membranes of TAL. The results of these studies are intended to provide a molecular basis for understanding the function of this important secretory KATP channel and it's regulation in health and disease.
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