The plasma membrane (sarcolemma) of cardiac myocytes has high levels of Na+-Ca2+ exchange activity. Na+-Ca2+ exchange is an important regulator of intracellular Ca2+ and thus a major determinant of myocardial contractility. Na+-Ca2+ exchange is upregulated and may take on added significance during hypertrophy and heart failure. It is important to investigate the structure and molecular properties of the cardiac Na+-Ca2+ exchange molecule to gain understanding of the role of the exchanger in physiology and pathophysiology. Towards this goal, the specific aims of the proposal are as follows: 1. Structure: Helix Packing. The Na+-Ca2+ exchanger has nine transmembrane segments (TMSs). The objective is to determine the packing arrangement of these TMSs within the plasma membrane. The approach will primarily use a combination of cysteine mutagenesis in conjunction with crosslinking techniques. 2. Mechanism and Regulation. There are five parts to this aim: a. Charge movements. To determine residues involved in charge movements associated with ion translocation by the exchanger. Initial experiments will use a chimera approach. b. Mechanism of inactivation. To test the hypothesis that TMS 2 has a key role in Na+-dependent inactivation. Na+-dependent inactivation is a key regulator of Na+-Ca2+ exchange activity. c. Role of the first reentrant loop in exchanger function. To determine the importance of the structure of a reentrant loop in regulation and transport by the exchanger. d. Kinetics of Ca2+ binding and Ca2+-induced conformational changes. To determine the kinetics of the binding of regulatory Ca2+ and the subsequent conformational change. The exchanger transports Ca2+ but is also regulated by Ca2+ at a high affinity regulatory site. e. Application of FRET to study the Na+-Ca2+ exchanger. To apply fluorescence resonance energy transfer (FRET) for monitoring conformational changes of the exchanger within a living cell. Exchangers will be labeled with variants of green fluorescent protein (GFP).
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