Positively charged cyclic hexapeptides, novel blockers for the cardiac sarcolemma Na(+)-Ca2+ exchanger.
Positively charged cyclic hexapeptides have been synthesized and tested for their effects on the cardiac sarcolemma Na(+)-Ca2+ exchange activities with a goal to identify a potent blocker. The cyclic hexapeptides, having the different amino acid sequence, contain two arginines (to retain a positive charge), two phenylalanines (to control hydrophobicity), and two cysteines (to form an intramolecular S-S bond). The effect of cyclic hexapeptides were tested on Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange, by measuring the 45Ca fluxes in the semi-rapid mixer or monitoring the calcium-sensitive dye Arsenazo III and voltage-sensitive dyes (Oxanol-V or Merocyanine-540). Seven cyclic hexapeptides inhibit Na(+)-Ca2+ exchange with a different potency (IC50 = 2-300 microM). Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) inhibits the Na+i-dependent 45Ca uptake (Na(+)-Ca2+ exchange) and Ca2+i-dependent 45Ca uptake (Ca(2+)-Ca2+ exchange) in the isolated cardiac sarcolemma vesicles with IC50 = 10 +/- 2 microM and IC50 = 7 +/- 3 microM, respectively. Interaction of FRCRCFa with a putative inhibitory site does not involve a "slow" binding (a maximal inhibitory effect is already observed after t = 1 s of mixing). The inside positive potential, generated by Na+o-dependent Ca2+ efflux, was monitored by Oxanol-V (A635-A612) or Merocyanine-540 (A570-A500). In both assay systems, FRCRCFa inhibits the Na(+)-Ca2+ exchange with IC50 = 2-3 microM, while a complete inhibition occurs at 20 microM FRCRCFa. The forward (Na+i-dependent Ca2+ influx) and reverse (Na+o-dependent Ca2+ efflux) modes of Na(+)-Ca2+ exchange, monitored by Arsenazo III (A600-A785), are also inhibited by FRCRCFa. The L-Arg4-->D-Arg4 substitution in FRCRCFa does not alter the IC50, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory potency. At fixed [Na+]i (160 mM) or [Ca2+]i (250 microM) and varying 45Cao (2-200 microM), FRCRCFa decreases Vmax without altering the Km. Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca2+ either for Na(+)-Ca2+ or Ca(2+)-Ca2+ exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding.
Khananshvili
D
D
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
Shaulov
G
G
Weil-Maslansky
E
E
Baazov
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D002474:Cell-Free System; D004220:Disulfides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D008954:Models, Biological; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000494:pharmacology; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D002412:Cations; D004220:Disulfides; D009842:Oligopeptides; D010455:Peptides; D010456:Peptides, Cyclic; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium