Abstract
The Na +-Ca 2+ exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca 2+ for 3 Na + ions entering the cell, regulating intracellular Ca 2+ levels and thereby contractility. Na +-Ca 2+ exchanger activity is regulated by intracellular Ca 2+, which binds to a region (amino acids 371–508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca 2+ activates the exchanger and removes Na +-dependent inactivation. The physiological role of intracellular Ca 2+ regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH 2- and CO 2H-termini of the exchanger Ca 2+ binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca 2+ concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca 2+ concentrations. Titrations of Ca 2+ concentration versus FRET efficiency indicate a K D for Ca 2+ of ∼140 nM, which increased to ∼400 nM in the presence of 1 mM Mg 2+. Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca 2+ on a beat-to-beat basis during excitation-contraction coupling.
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