Published online before print November 18, 2004, doi: 10.1161/01.RES.0000151334.48676.68
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Submitted on January 16, 2004
Revised on October 15, 2004
Accepted on November 9, 2004
Cardiac Sodium-Calcium Exchanger Is Regulated by Allosteric Calcium and Exchanger Inhibitory Peptide at Distinct Sites
Christoph Maack ;
From the Johns Hopkins University, Institute of Molecular Cardiobiology, Division of Cardiology, Baltimore, Md.
* To whom correspondence should be addressed. E-mail: bor{at}jhmi.edu.
The sarcolemmal Na+-Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism in cardiac myocytes and thus essential for the regulation of Ca2+ homeostasis and contractile function. A cytosolic region (f-loop) of the protein mediates regulation of NCX function by intracellular factors including inhibition by exchanger inhibitory peptide (XIP), a 20 amino acid peptide matching the sequence of an autoinhibitory region involved in allosteric regulation of NCX by intracellular Na+,Ca2+ and phosphatidylinositol-4,5-biphosphate (PIP2). Previous evidence indicates that the XIP interaction domain can be eliminated by large deletions of the f-loop that also remove activation of NCX by intracellular Ca2+. By whole-cell voltage clamping experiments, we demonstrate that deletion of residues 562 to 679, but not 440 to 456, 498 to 510, or 680 to 685 of the f-loop selectively eliminates XIP-mediated inhibition of NCX expressed either heterologously (HEK293 and A549 cells) or in guinea pig cardiac myocytes. In contrast, by plotting INCX against reverse-mode NCX-mediated Ca2+ transients in myocytes, we demonstrate that Ca2+-dependent regulation of NCX is preserved in 
562 to 679, but significantly reduced in the other three deletion mutants. The results indicate that residues 562 to 679 are critically involved in the inhibitory action of XIP, but are not implicated in Ca2+-dependent regulation of NCX. Vice versa, amino acids 440 to 456, 498 to 510, and 680 to 685 are involved in regulation of NCX by Ca2+, but not XIP. We conclude that residues 562 to 679 contain the regulatory site for XIP, but not Ca2+. This finding may facilitate investigation of XIP-mediated regulation of NCX by cellular factors.
Key words: cardiac myocytes • adenovirus • mutation
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