Identification of Important Amino Acid Residues of the Na + -Ca 2+ Exchanger Inhibitory Peptide, XIP

Abstract

The Na+-Ca2+ exchanger plays an important role in cardiac contractility by moving Ca2+ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na+-Ca2+ exchange activity were determined. Exchange activity was assessed as 45Ca2+ uptake into Na+-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function.

Local PIP2 signals: when, where, and how?

“…The list of agents employed includes many different cationic peptides and the aminoglycosides. The cationic peptides include two putatively specific inhibitors of NCX1, the "XIP" peptide that represents a sequence from the exchanger itself [32], and a cyclic cationic peptide found to be a strong exchanger inhibitor [45]. These peptides inhibit the PIP 2 -sensitive KATP channels almost equally powerfully as they inhibit Na/Ca exchange (not shown), suggesting that PIP 2 -binding may indeed play a role.…”

The role of the Na+/Ca2+ exchanger (NCX) in neurons following ischaemia

“…XIP is an autoregulatory region of the intracellular loop of the NCX protein that effectively inhibits NCX activity. [41][42][43][44] Exogenous XIP can act as a specific NCX inhibitor. Intracellular sodium binds to the XIP region and induces NCX inactivation, while increasing intracellular calcium, via a separate binding site, promotes NCX activation by alleviating sodium inactivation.…”

Sodium/Calcium Exchanger: Influence of Metabolic Regulation on Ion Carrier Interactions

“…Mutations that eliminate inactivation produce stronger interactions between phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] and XIP, whereas those interactions are weakened by mutations that promote inactivation (116,176). Mutations in the XIP peptide indicate that although its full length is required for maximal potency, the more relevant inhibitory components are basic and aromatic residues between positions 5 and 16 (117). A recent work claims that in NCX the binding site for XIP may be located in the loop zone between amino acids 562 and 679 (171); at any rate, although the site of interaction of the XIP region with the rest of the NCX molecule has not been identified, it has been suggested that this region functions as an auto inhibitory domain that shifts the active-inactive states of the Na ϩ /Ca 2ϩ exchanger.…”