AN OXONOL DYE IS THE MOST POTENT KNOWN INHIBITOR OF BAND 3-MEDIATED ANION-EXCHANGE

When cells are acutely exposed to the oxonol dye, bis(1,3-dibutylbarbi turic acid)pentamethine oxonol (diBA), at O degrees C, the concentrati on that gives half inhibition of Cl- exchange (IC50) is 0.146 +/- 0.01 3 mu M (n = 12) initially, but the inhibition increases with time. The se characteristics indicate that a rapid initial binding is followed b y a slow conformational change that makes the binding tighter. If diBA is allowed to equilibrate with band 3, the IC50 is only 1.05 +/- 0.13 nM (n = 5), making diBA a more potent inhibitor than 4,4'-diisothiocy anostilbene-2,2'-disulfonic acid (DIDS), for which the IC50 under simi lar conditions is 31 +/- 6 nM [T. Janas, P. J. Bjerrum, J. Brahm, and J. O. Wieth. Am. J. Physiol. 257 (Cell Physiol. 26): C601-C606, 1989]. Inhibition by diBA is very slowly reversible at 0 degrees C (t(1/2) > 50 h), but the effect is more readily reversible at higher temperatur es. DiBA competes with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) fo r inhibition, suggesting an external site of action. In contrast to DI DS and DNDS, however, increasing Cl- concentrations do not decrease th e inhibitory effect of diBA, indicating that the inhibition is not com petitive. Thus diBA may be useful for investigating conformational cha nges during anion exchange and for stopping transport without preventi ng substrate binding. However, when diBA and other oxonols are used to sense membrane potential, they may have undesirable side effects on a nion transport processes.