Human Rhesus-associated glycoprotein mediates facilitated transport of NH 3 into red blood cells

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Abstract
Rhesus (Rh) antigens are carried by a membrane complex that includes Rh proteins (D and CcEe), Rh-associated glycoproteins (RhAG), and accessory chains (LW and CD47) associated by noncovalent bonds. In heterologous expression systems, RhAG and its kidney orthologs function as ammonium transporters. In red blood cells (RBCs), it is generally accepted that NH 3 permeates by membrane lipid diffusion. We have revisited these issues by studying RBC and ghosts from human and mouse genetic variants with defects of proteins that comprise the Rh complex. In both normal and mutant cells, stopped-flow analyses of intracellular pH changes in the presence of inwardly directed methylammonium (CH 3 NH + 3 +CH 3 NH 2 ) or ammonium (NH + 4 +NH 3 ) gradients showed a rapid alkalinization phase. Cells from human and mouse variants exhibited a decrease in their kinetic rate constants that was strictly correlated to the degree of reduction of their RhAG/Rhag expression level. Rate constants were not affected by a reduction of Rh, CD47, or LW. CH 3 NH 2 /NH 3 transport was characterized by ( i ) a sensitivity to mercurials that is reversible by 2-mercaptoethanol and ( ii ) a reduction of alkalinization rate constants after bromelain digestion, which cleaves RhAG. The results show that RhAG facilitates CH 3 NH 2 /NH 3 movement across the RBC membrane and represents a potential example of a gas channel in mammalian cells. In RBCs, RhAG may transport NH 3 to detoxifying organs, like kidney and liver, and together with nonerythroid tissue orthologs may contribute to the regulation of the systemic acid–base balance.