Arginine (Arg) is the sole substrate for nitric oxide synthase (NOS) activity to produce nitric oxide (NO), a signaling molecule which is crucial for many physiologic and pathologic processes. Arg is not synthesized by cardiac myocytes and must be imported from the plasma. Thus, the membrane-bound carrier protein(s) responsible for Arg transport may play a role as important as Arg itself in the pathophysiology of the Arg-NO system. Our preliminary data in cardiac myocytes show large Arg-activated currents whose properties are consistent with the low-affinity cationic amino acid transporter CAT-2A. Our data also show a) the presence of CAT-2A mRNA transcripts in cardiac myocytes, b) Arg-dependent transient charge movements, which allow detailed kinetic studies on FM-dependent Arg transport, c) Arg-activated NO release, and d) NO inhibition of Arg currents, suggesting acute regulation of Arg transport by this signaling molecule. The goal of this project is to identify and characterize this cardiac arginine transporter, quantitatively solve its kinetic mechanism, and study its potential regulation by NO. To achieve this goal, Arg transport and the carrier protein will be studied with a combination of molecular biological, biochemical, fluorescence, and electrophysiological techniques to investigate the hypothesis that Arg-activated currents in cardiac myocytes are produced by the low-affinity CAT-2A transporter. Proposed experiments will characterize this transporter electrophysiologically and biochemically by determining substrate specificities, apparent affinities, sensitivity to inhibitors, and the Vm dependence of Arg transport. Experiments will also take advantage of our preliminary data showing Arg-dependent charge movements to solve the kinetic reaction scheme that describes Arg transport. Finally, experiments will study inhibition of Arg-activated currents by NO as well as NO-sensitive protein kinase-mediated phosphorylation of the transporter to solve the mechanism by which NO acutely regulates Arg transport in cardiac myocytes. Altogether, these studies will provide a detailed picture of the molecular events that take place during cationic amino acid transport into cells and how regulatory mechanisms may alter transport function.
