The carrier reorientation step in erythrocyte choline transport: pH effects and the involvement of a carrier ionizing group

Abstract

Under zero-trans conditions, the facilitated transport of choline across the erythrocyte membrane is limited by the rate of reorientation of the free carrier; as a result the pH dependence of this step can be investigated, independent of other steps in transport. It is found that as the pH declines (between 8.0 and 6.0) the rate of inward movement of the free carrier rises and the rate of outward movements falls, so that the partition of the free carrier increasingly favors the inward-facing form. When the pH of the cell interior and of the medium are varied independently, the partition responds to the internal but not the external pH. The membrane potential, which varies somewhat as the pH is altered, has no effect on the carrier partition. The analysis of the results indicates that the carrier mobility is dependent on an ionizing group of pKa 6.8, which is exposed on the cytoplasmic surface of the membrane in the inward-facing carrier; in the outward-facing carrier the ionizing group appears to be masked, in that its pKa is shifted downward by more than one unit. The observations can be explained by assuming that an ionizing group is located in the wall of a gated channel connecting the substrate site with the cytoplasmic face of the cell membrane.