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* Cardiovascular Research Laboratories, and Departments of Physiology and Medicine (Cardiology), David Geffen School of Medicine at the University of California, Los Angeles, California 90095-1760
Correspondence: Address reprint requests to Kenneth D. Philipson, MRL 3-465, Cardiovascular Research Laboratories, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA 90095-1760. Tel.: 310-825-7679; E-mail: kphilipson{at}mednet.ucla.edu.
The Na+-Ca2+ exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca2+ for 3 Na+ ions entering the cell, regulating intracellular Ca2+ levels and thereby contractility. Na+-Ca2+ exchanger activity is regulated by intracellular Ca2+, which binds to a region (amino acids 371–508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca2+ activates the exchanger and removes Na+-dependent inactivation. The physiological role of intracellular Ca2+ regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH2- and CO2H-termini of the exchanger Ca2+ binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca2+ concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca2+ concentrations. Titrations of Ca2+ concentration versus FRET efficiency indicate a KD for Ca2+ of 
140 nM, which increased to 400 nM in the presence of 1 mM Mg2+. Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca2+ on a beat-to-beat basis during excitation-contraction coupling.
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