Conformational changes of the Ca2+ regulatory site of the Na+-Ca2+ exchanger detected by FRET

Abstract

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.