Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 2. Mechanism of inhibition by bepridil

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Abstract
Bepridil, an antiarrhythmic agent, inhibits Na-Ca exchange in cardiac sarcolemmal membrane vesicles (Ki = 30 .mu.M) by a novel mechanism, different from that determined for amiloride analogues [Slaughter, R.S., Garcia, M. L., Cragoe, E. J., Jr., Reeves, J. P., and Kaczorowski, G. J. (1988) Biochemistry (preceding paper in this issue)]. Bepridil causes partial inhibition of Nai-dependent Ca2+ uptake but complete block of Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange is noncompetitive vs Ca2+ but competitive vs Na+ in both K+ and sucrose. Bepridil also blocks Ca-Ca exchange, with or without K+ present. However, K+ has two effects on inhibition: it reduces the potency of bepridil and causes inhibition to become partial. Inhibition of Ca-Ca exchange displays noncompetitive kinetics vs Ca2+ in either sucrose or K+. Dixon analyses of Na-Ca exchange inhibition caused by mixtures of bepridil and amiloride analogues demonstrated that these compounds produce a competitive interaction at a common carrier site that is evident only at low concentrations of amiloride inhibitors. Hill plots of bepridil inhibition of Na-Ca and Ca-Ca exchange display unitary Hill coefficients. These results indicate that bepridil interacts at only one substrate-binding site, the site selective for Na+, where amiloride analogues also preferenially interact. However, unlike amiloride, bepridil does not interact at the common Na+, Ca2+-binding site of the carrier. During stimulation of Ca-Ca exchange, K+ may bind at other sites besides the site selective for Na+, because in addition to being competitive with bepridil, it prevents complete abolition of Ca-Ca exchange, suggesting that this Na+ site is not involved in carrier turnover. These findings indicate that bepridil is a mechanism-based Na-Ca exchange inhibitor that interacts at a transporter site which binds Na+ but not Ca2+.