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 Ca²⁺-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 μM). Phe-Arg-Cys-Arg-Cys- Phe-CONH₂ (FRCRCFa) inhibits the Na⁺(i)-dependent ⁴⁵Ca uptake (Na⁺- Ca²⁺ exchange) and Ca²⁺(i)-dependent ⁴⁵Ca uptake (Ca²⁺-Ca²⁺ exchange) in the isolated cardiac sarcolemma vesicles with IC₅₀ = 10 ± 2 μM and IC₅₀ = 7 ± 3 μM, 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 μM, while a complete inhibition occurs at 20 μM 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 μM) and varying ⁴⁵Ca(o) (2- 200 μM), FRCRCFa decreases V(max) without altering the K(m). Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca²⁺ either for Na⁺-Ca²⁺ or Ca²⁺-Ca²⁺ exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding.