Abstract
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.
Highlights
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
We recently found that in the cardiac sarcolemma vesicles the Phe-Met-Arg-Phe-CONH2 (FMRFa)l tetrapeptide and its analogs yield a complete inhibition of Na" _Ca2+ and Ca 2+_ Ca2+ exchanges, exhibiting IC so = 10-6_10-3 M [23]
FRCRCFa-induced Inhibition ofNa+,dependent Ca2 + Influx and Na +a-dependent Ca2 + Efflux, Monitored by Arsenazo IIISince the Na+-Ca2+ exchange can operate in forward (Na\-dependent Ca2+ influx) and reverse (Na+o·dependent Ca2+ efflux) modes, the effect of20 !LM FRCRCFa was tested on both exchange modes
Summary
Vol 270, No 27, Issue of July 7, pp. 16182-16188, 1995 Printed in U.S.A. Positively Charged Cyclic Hexapeptides, Novel Blockers for the Cardiac Sarcolemma Na+ -Ca2 + Exchanger*. 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. RFa-like peptides and opiate agonists and antagonists are mutually exclusive inhibitors of Na +_Caz+ exchange, suggesting that they may bind to the same site [23]. This putative "opiate-like" site lacks the pharmacological properties of known opiate receptors and may be located on the exchanger or at its vicinity [23]. Linear peptide inhibitors attribute common structural disadvantages, which seem to be difficult to overcome without application of alternative approaches Both the XIP and FMRFa peptides contain positively charged amino acids Arg and/or Lys, which make them attractive for proteolytic enzymes. The present findings may be an attractive starting point for design of even better inhibitors of the N a +Caz+ exchanger
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