The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: II. Kinetic properties of NBD-taurine transfer in asymmetric conditions

Abstract

The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facingE _i or an outward-facingE _o, conformation, each capable of binding either a monovalent anion or a divalent anion+a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [E _i], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4′-dinitro-2,2′-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions.