(KATP) channels are participants in the response of the heart and vascular system to change of metabolic state, including ischemia. The proposed study will examine the structural basis of KATP channel regulation, by combined molecular biological, biochemical, and electrophysiological experiments. Recently, the principal investigator cloned the high affinity sulfonylurea receptor (SUR) and reconstituted KATP channel activity as a complex of the SUR with a small inward rectifier subunit (Kir6.2). This major advancement allows him to begin to dissect the structural basis of KATP channel regulation. The investigators hypothesize that nucleotide regulation of the channel results from binding to the SUR, and have isolated SUR mutations that interfere with nucleotide regulation. In order to extend their preliminary data, four experimental series are proposed, addressing the following specific questions: (1) How do intracellular nucleotides inhibit KATP channels? (2) How do cytoplasmic nucleotides activate KATP channels? (3) What are the structural requirements for functional interaction of SUR and Kir6.2 subunits? (4) Can one exogenously express a functional cardiac KATP channel complex? Nucleotide inhibition of mutant and chimeric KATP channels will be examined in inside-out membrane patches in order to localize structural domains responsible for nucleotide regulation, and for interaction between SUR and Kir6.2 subunits. Photo-affinity labeled nucleotides will be used to determine nucleotide binding specificity of the SUR. By homology screening, they will use the cloned pancreatic SUR and Kir6.2 subunits to clone the necessary components to reconstitute cardiac KATP channel activity. Work on this project to date has contributed substantially to the current understanding of the role and regulation of cardiac KATP channels. The results of the proposed experiments will bring detailed insight into the fundamental structural features responsible for ligand regulation of ion channels generally, and of KATP channels specifically. The work will provide information that will ultimately underlie the development of rational therapies for the treatment of cardiac ischemia and arrhythmias.
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