Bicarbonate exchange through the human red cell membrane determined with [14C] bicarbonate.

Abstract

Bicarbonate transport across human red cell membranes was studied between 0 and 10.degree. C at alkaline pH values by determining efflux of 14C-labeled bicarbonate from resealed erythrocyte ghosts. Transfer of labeled CO2 was eliminated as a source of error, when formation of intracellular 14CO2 was inhibited with carbonic anhydrase inhibitors. There were no fundamental differences between characteristics of bicarbonate and of Cl self-exchange as was inferred from previous studies of Cl-bicarbonate exchange. Efflux of radioactivity could be reduced more than 99% by reversible and irreversible inhibitors of anion transport. Inhibition of Cl and bicarbonate self-exchange was linearly related to binding of 4,4''-diisothiocyanostilbene-2,2''-disulphonic acid (DIDS) to the membranes. Complete (i.e., > 99%) inhibition was obtained after binding of 1.2 .times. 106 DIDS molecules/cell. Bicarbonate self-exchange proved a saturable function of bicarbonate concentration, with a maximum at external and internal concentrations of .apprx. 100 mM, showing self-depression at higher bicarbonate concentrations and half-maximum exchange flux at a concentration of 10 mM. Results were consistent with the hypothesis that the exchange mechanism had 2 anion binding sites, 1 mediating ion transport and the other transport inhibition. Maximum exchange flux of bicarbonate was about 30% larger than that of Cl, and affinity of bicarbonate for the transport site was about 3 times larger than that of Cl. Apparent activation energy of bicarbonate exchange was 28 kcal/mol, the same order of magnitude as found for other inorganic anions between 0 and 10.degree. C. Ability of other inorganic anions to exchange with bicarbonate decreased in the sequence Cl > NO3 > F > Br .gtoreq. I, corresponding to the sequence of rate of self-exchange of halides. Counter-transport of bicarbonate could be driven by a Cl gradient, when ghosts containing KCl were suspended in a medium containing traces of labeled bicarbonate in addition to a non-permeating anion. Concentration ratios (ci/co) up to about 1000 could be obtained. Bicarbonate was transported by the inorganic anion exchange mechanism of the erythrocyte membrane. Slight differences between the exchange kinetics of Cl and bicarbonate were explained by differing affinities of the 2 anions for the 2 anion binding sites of the transport system.