Localization of an exchange inhibitory peptide (XIP) binding site on the cardiac sodium-calcium exchanger

The exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is a potent inhibitor of cardiac Na-Ca exchange activity, This study attempted to identify the XIP binding site on the Na-Ca exchange protein. Bovine cardiac sarcolemmal vesicles were proteolyzed and fractionated by XIP-affinity column chromatography, A 24 kDa fragment was purified and subjected to amino acid sequence analysis. A negatively charged region of intracellular loop f of the Na-Ca exchange protein (IDDDIFEEDEN; aa 445-455) was identified, The affinity and specificity of XIP interaction with peptides IDDDIFEEDEN and GEDDDDDECGEE (another negatively charged region of the Na-Ca exchange protein) were examined. XIP cross-linked to peptide IDDDIFEEDEN but not GEDDDDDECGEE in a pH-dependent manner, Fluorescence titration binding studies indicated that binding of IDDDIFEEDEN with XIP was saturable (Kd=5 mu M) while binding with GEDDDDDECGEE was not specific, These data suggest that amino acids 445-455 on Na-Ca exchange loop fare involved in XIP binding. (C) 1997 Academic Press.