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

The kinetics of human red blood cell Cl transport have been 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'- stilbene disulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, we have estimated the number of Cl ions transported during the putative outward recruitment. Resealed red cell ghosts containing only 70 muM 36Cl were resuspended at 0 degrees C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, or approximately 10(6) Cl ions per ghost, followed by a much slower further loss of Cl. The rapid loss of 10(6) Cl ions per 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 first half- turnover of the transport cycle. These data are strong evidence that the influx and efflux events in the catalytic cycle for anion transport do not take place simultaneously, and that the stoichiometry of the transport cycle is close to one pair of anions exchanged per band 3 monomer.