MOLECULAR MECHANISMS OF KIDNEY KATP CHANNEL FUNCTION

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DESCRIPTION (Adapted from the Applicant's Abstract): Potassium homeostasis is ultimately dependent on unique ATP-sensitive K channels, KATP, in the renal cortical collecting duct. These channels provide the final regulated step in kidney's potassium secretory process. They have recently discovered that functionally similar low-sulfonylurea affinity KATP channels are formed by two heterologous molecules, products of Kir1.1a and CFTR genes. With observations that the expression patterns of Kir1.1a and CFTR overlap in the distal nephron along the apical membrane, our discovery offers compelling evidence that the native potassium secretory KATP channels are comprised of Kir1.1a and CFTR. As a logical progression the present proposal is focused on elucidating the molecular mechanisms responsible for secretory K channel function. Specifically, a stepwise multidisciplinary approach, combining biochemical techniques, electrophysiology, molecular genetics, and transgenic technology, will be employed to develop a molecular understanding of the following critical aspects of channel function and modulation: 1) Our hypothesis that the quatemary structure of the CFTR/Kir1.1a complex is comprised of a Kir1.1a tetramer surrounded by a variable number of CFTR subunits, will be critically tested by studying the biophysical properties of Kirl.la concatamers and CFTRCKirl.la fusion proteins that have a fixed stoichiometry; 2) The relative functional roles of the three different nucleotide binding domains (NBD) in Kir1.1a/CFTR will be accessed by studying the functional consequences of loss and gain-of-function mutations in the each NBD; 3) Having identified one broad intersubunit interaction domain with the two-hybrid system in yeast, we will continue to delimit this region and map others with this system. Elucidation of protein-protein interaction domains of CFTR and Kirl.la should provide necessary information to understand how CFTR confers AlT and Sulfonylurea sensitivity on Kir1.1a; 4) The role of these domains will be critically evaluated at the functional level using patch clamp analysis on chimeric and mutated channels which have altered or implanted interaction sites; 5) Finally these observations in the recombinant channels will be applied back to the native K secretory channel. Specifically, the properties of the native channel will be extensively characterized in the CFTR knock out mouse by patch-clamp analysis to determine if CFTR is the sole, or even major, ATP binding cassette component of the native secretory channel. These studies represent a timely and important extension of the principal investigator's work, and should ultimately provide considerable insight into the basis of renal K handling and K homeostasis in health and disease.