Energy metabolism enables cells to maintain an internal milieu different from that of the environment. It is no wonder, then, that eukaryotic cells have evolved mechanisms whereby changes in energy metabolism feed back upon and regulate ion transport. The applicant has recently discovered two such mechanisms in mammalian cardiac cells, where they may alter excitability and excitation-contraction coupling during ischemia. The first involves the regulation of L-type calcium channels. He has found that sudden jumps in intracellular MgATP (but not in either Mg2+ or ATP4- alone) up-regulate the calcium current in ventricular myocytes. This response is not dependent on phosphorylation: nonhydrolyzable ATP analogs support the response, and it remains robust even in the presence of protein kinase inhibitors. The evidence suggests a direct allosteric effect of MgATP on calcium channels. This application seeks to elucidate the biophysical and structural bases of this effect using electrophysiological and molecular genetic approaches. The second mechanism focuses on oscillations of membrane current and Ca2+ transients in which energy metabolism is the primary oscillator. The aplicant has found that heart cells subjected to moderate, reversible metabolic stress (for example, removal of external glucose) undergo spontaneous oscillations of membrane current. Large outward currents develop gradually, peak and subside with a period of one-two min; the currents are blocked by glibenclamide and suppressed by glucose. Although such oscillations are not initiated by intracellular Ca2+, depolarization-evoked Ca2+ transients are inhibited as the outward currents peak. Previous work in cardiac cystosolic extracts and in intact cells has demonstrated that glycolytic metabolites oscillate spontaneously, due to opposing feedback effects on the rate- limiting enzyme phosphofructokinase. In line with this idea, the investigator's preliminary data reveal that the membrane current oscillations can be initiated (or their phase reset) by sudden liberation of intracellular ADP or ATP using flash photolysis of caged compounds. The applicant will test the hypothesis that the membrane current is carried by ATP-dependent potassium channels driven by primary oscillations in the rate of glycolysis. Intracellular NADH and ATP concentrations will be measured to verify directly the presence of metabolic oscillations and to determine their magnitude. The investigator will also investigate the basis of the concomitant changes in excitation-contraction coupling. These two pathways by which metabolism can alter ionic currents merit investigation not only because of their novelty but also because of their potentially important contributions to arrhythmias and disorders of contractility.
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