Rapid exchange of fluoroethylamine via the Rhesus complex in human erythrocytes: 19F NMR magnetization transfer analysis showing competition by ammonia and ammonia analogues

Abstract

A remarkable recent discovery in red blood cell function is that the Rhesus antigen complex that for so long was considered to be simply a means of cell recognition is also the ammonia transporter. It catalyzes transmembrane exchange of ammonia on the subsecond time scale, and yet because of a lack of rapid-exchange methodology its kinetics had not been characterized. The flux of ammonia varies appreciably in diverse clinical states, and a convenient method for its characterization would be of basic and of clinical diagnostic value. Fluoroethylamine is water-soluble and when added to a suspension of human red blood cells (RBCs) displays the experimentally useful property of giving separate 19F NMR spectral peaks for the populations inside and outside the cells. By using two-site, one-dimensional magnetization exchange spectroscopy (1D-EXSY), the transmembrane exchange of fluoroethylamine was measured; it was found to occur on the subsecond time scale with an apparent first-order rate constant for efflux, under the equilibrium exchange conditions, of 3.4 s(-1). The method was used to characterize the concentration, temperature, and pH dependence of the exchange rate constant. We determined the extent of competitive inhibition exhibited by ammonia and two molecules that contain an amine group (ethylamine and methylamine). Inhibition of the exchange by incubating the suspension with anti-RhAG antibody, and no inhibition by anti-RhD antibody, suggested specificity of exchange via the RhAG protein of the Rh complex.