The sarcolemmal Na+-Ca2+ exchanger is the dominant Ca2+ efflux mechanism in myocardial cells. Thus, the exchanger has a critical role in the regulation of myocardial contractility. Molecular knowledge of this essential transporter will help understand both normal and diseased hearts. The long-term objective of this research is to elucidate the structure and function of the Na+-Ca2+ exchanger to better understand the mechanism and regulation of transport. Toward this goal, specific aims are as follows: 1. Ion translocation. The pathway for the movement of Na+ and Ca2+ through the exchange protein will be determined. Cysteine-scanning mutagenesis will be initiated to analyze the importance of amino acids residues in transmembrane segments hypothesized to be important in ion translocation. Systematic analysis of the effects of sulfhydryl reagents on cysteine mutants will help determine residue accessibility and will provide information on the structure of the translocation pathway (e.g., pore width and depth; alpha-helicity). Further mutagenesis of aromatic residues, hydroxyl-containing residues, and residues conserved among exchangers is also planned. Mutants will be expressed in Xenopus oocytes and assessed by measurements of 45Ca2+ fluxes and exchange currents. 2. Regulation. The Na+-Ca2+ exchanger is subject to diverse and complex regulation. For example, the exchanger is regulated by the binding of nontransported Ca2+ to a high affinity intracellular site. In addition, the exchanger has an autoregulatory region, has an ankyrin-binding site, and is stimulated by anionic phospholipids such as PIP2. The molecular bases of these phenomena will be determined using a combination of protein biochemistry, molecular biology, and electrophysiology. 3. Structure. Aspects of the secondary and tertiary structure of the Na+-Ca2+ exchange protein will be determined. Topology will be assessed by a variety of techniques. A project using a combination of cysteine mutagenesis and protein biochemistry is also being initiated to determine the packing of alpha- helices within the membrane.
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