Cardiac sodium-calcium exchanger is regulated by allosteric calcium and exchanger inhibitory peptide at distinct sites

The sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) is the main Ca²⁺ extrusion mechanism in cardiac myocytes and is 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 phosphatidylinosito]-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 562-679, but not 440-456, 498-510, or 680-685 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 I_NCX against reverse-mode NCX-mediated Ca²⁺ transients in myocytes, we demonstrate that Ca ²⁺-dependent regulation of NCX is preserved in Δ562-679, but significantly reduced in the other three deletion mutants. The findings indicate that f-loop residues 562-679 may contain the regulatory site for endogenous XIP, but this site is distinct from the Ca²⁺-regulatory domains of the NCX. Because regulation of the NCX by Na⁺ and PIP₂ involves the endogenous XIP region, the Δ562-679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.