Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS.

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.