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 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 562-679, 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 Ca2+-dependent regulation of NCX is preserved in Delta562-679, 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 Delta562-679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.
Maack
Christoph
C
Johns Hopkins University, Institute of Molecular Cardiobiology, Division of Cardiology, Baltimore, Md 21205-2195, USA.
Ganesan
Anand
A
Sidor
Agnieszka
A
O'Rourke
Brian
B
D000200:Action Potentials; D000230:Adenocarcinoma / Q000473:pathology; D000256:Adenoviridae / Q000235:genetics; D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002460:Cell Line / Q000378:metabolism; D045744:Cell Line, Tumor / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008175:Lung Neoplasms / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D012508:Sarcolemma / Q000378:metabolism; D017384:Sequence Deletion; D012964:Sodium / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000502:physiology
D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C066745:exchanger inhibitory peptide; D012964:Sodium; D002118:Calcium