The mitochondrial KATP channel (mitoKATP) regulates matrix volume. Matrix volume, in turn, is hypothesized to control the activation state of electron transport. MitoKATP opens and closes in response to cellular levels of ATP, GTP and long-chain acyl-CoA esters. It is also a pharmacological receptor for K+ channel openers and the antidiabetic sulfonylureas. The broad objectives of this project are (1) to understand the molecular mechanisms of regulated K+ flux through mitoKATP; and (2) to understand the metabolic signalling role of mitoKATP activity in E. coli and/or S. cerevisiae; (3) to elucidate the molecular regulatory mechanisms of native and mutagenized mitoKATP; and (4) to test hypotheses relating to the functional role of mitoKATP. Peptide sequence for the sulfonylurea receptor subunit will be obtained and used to isolate its cDNA. Development of an expression system will be followed by protein isolation, reconstitution and assay of regulated K+ flux. K+ flux, measured with a K+ -specific fluorescent probe, will be assayed for inhibition by ATP, palmitoyl CoA, and glyburide, and reversal of inhibition by GTP and K+ channel openers. K+ flux will also be measured in intact mitochondria in order to examine the physiological role of mitoKATP using control analysis.
