Stoichiometry of a half-turnover of band 3, the chloride transport protein of human erythrocytes.

Abstract

The kinetics of human red blood cell Cl transport were studied under nonequilibrium conditions to determine whether or not an outward Cl gradient can recruit the transport protein from an inward-facing to an outward-facing configuration. Three kinds of evidence are consistent with this outward recruitment. First, the initial net Cl efflux into a Cl-free phosphate medium is independent of the intracellular Cl concentration in the range 20-170 mM. Second, an outward Cl gradient strongly enhances the inhibitory potency of DNDS (4,4''-dinitro-2,2''-stilbenedisulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, the number of Cl ions transported during the putative outward recruitment was estimated. Resealed red cell ghosts containing only 70 .mu.M 36Cl were resuspended at 0.degree. C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, .apprx. 106 Cl ions leave each ghost, followed by a much slower further loss of Cl. The rapid loss of 106 Cl ions/ghost, which is abolished by pretreatment with DIDS (4,4''-diisothiocyano-2,2''-stilbene disulfonate), appears to represent the Cl that is transported during the 1st half-turnover of the transport cycle. The influx and efflux events in the catalytic cycle for anion transport apparently do not take place simultaneously. The stoichiometry of the transport cycle evidently is close to 1 pair of anions exchanged per band 3 monomer.