VOLTAGE-DEPENDENCE OF DIDS-INSENSITIVE CHLORIDE CONDUCTANCE IN HUMAN RED-BLOOD-CELLS TREATED WITH VALINOMYCIN OR GRAMICIDIN

Net K and Cl effluxes induced by valinomycin or by gramicidin have bee n determined directly at varied external K, denoted by [K](o), in the presence and absence of the anion transport inhibitors DIDS (4,4'-diis o-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analog ue SITS -acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 mu M DIDS, or 100 mu M SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K](o), by only 59-6 7%. This partial inhibition by 10 mu M DIDS at 1 mM [K](o) remains con stant when the concentration of DIDS, or when the temperature or pH du ring pretreatment with DIDS, are increased. Observations of such parti al inhibition previously prompted the postulation of two Cl conductanc e pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K](o) corresponding to values of E(K) between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being less than or equal to 0.1 mu Mol/g Hb/min , both with valinomycin (1 mu M) and with gramicidin (0.06 mu g/ml). A t lower [K](o), where E(K) is below similar to -35 mV, the DIDS-insens itive fraction of net Cl efflux increases to 2.6 mu Mol/g Hb/min with valinomycin (1 mu M), and to 4.8 mu Mol/g Hb/min with gramicidin (0.06 mu g/ml). With net fluxes determined from changes in mean cell volume , and with membrane potentials measured from changes in the external p H of unbuffered red cell suspensions, a current-voltage curve for DIDS -insensitive Cl conductance has been deduced. While specific effects o f varied [K](o) on net Cl efflux are unlikely but cannot strictly be r uled out, the results are consistent with the hypothesis that DIDS-ins ensitive Cl conductance turns on at an E(m) of similar to -40 mV.