KINETICS OF RESIDUAL CHLORIDE TRANSPORT IN HUMAN RED-BLOOD-CELLS AFTER MAXIMUM COVALENT 4,4'-DIISOTHIOCYANOSTILBENE-2,2'-DISULFONIC ACID-BINDING

Irreversible inhibition, 99.8% of control values for chloride transpor t in human red blood cells, was obtained by well-established methods o f maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfo nic acid (DIDS). The kinetics of the residual chloride transport (0.2% , 106 pmol.cm-2.s-1) at 38-degrees-C, pH 7.2) was studied by means of Cl-36- efflux. The outside apparent affinity, expressed by K1/2,c(o), was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Border s, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemi stry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of t he residual chloride flux depended on the extracellular chloride conce ntration, but was not purely competitive. The half-inhibition concentr ations at [Cl(o)] = 150 mM in control cells (K(i,o)) and covalently DI DS-treated cells (K(i,c)) were: DIDS, K(i,c) = 73 nM; DNDS, K(i,o) = 6 .3 muM, K(i,c) = 22 muM; phloretin, K(i,o) = 19 muM, K(i,c) = 17 muM; salicylate, K(i,o) = 4 mM, K(i,c) = 8 mM; Killer III, K(i,o) = 10 muM, K(i,c) = 10 muM.