Effects of membrane potential on electrically silent transport. Potential-independent translocation and asymmetric potential-dependent substrate binding to the red blood cell anion exchange protein

Abstract

Tracer anion exchange flux measurements have been carried out in human red blood cells with the membrane potential clamped at various values with gramicidin. The goal of the study was to determine the effect of membrane potential on the anion translocation and binding events in the catalytic cycle for exchange. The conditions were arranged such that most of the transporters were recruited into the same configuration (inward-facing or outward-facing, depending on the direction of the Cl- gradient). We found that the membrane potential has no detectable effect on the anion translocation event, measured as 36Cl(-)-Cl- or 36Cl(-)-HCO3- exchange. The lack of effect of potential is in agreement with previous studies on red cells and is different from the behavior of the mouse erythroid band 3 gene expressed in frog oocytes (Grygorczyk, R., W. Schwarz, and H. Passow. 1987. J. Membr. Biol. 99:127-136). A negative potential decreases the potency of extracellular SO4= as an inhibitor of either Cl- or HCO3- influx. Because of the potential-dependent inhibition by SO4=, conditions could be found in which a negative intracellular potential actually accelerates 36Cl- influx. This effect is observed only in media containing multivalent anions. The simplest interpretation of the effect is that the negative potential lowers the inhibitory potency of the multivalent anion by lowering its local concentration near the transport site. The magnitude of the effect is consistent with the idea that the anions move through 10-15% of the transmembrane potential between the extracellular medium and the outward-facing transport site. In contrast to its effect on extracellular substrate binding, there is no detectable effect of membrane potential on the competition between intracellular Cl- and SO4= for transport sites. The lack of effect of potential on intracellular substrate binding suggests that the access pathway leading to the inward-facing transport site is of lower electrical resistance than that leading to the extracellular substrate site.