Structural determinants of NH3 and NH4+ transport by mouse Rhbg, a renal Rh glycoprotein.

Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH₃/NH₄⁺ transport. The structure of Rhbg is not yet resolved; however, a high-resolution crystal structure of AmtB, a bacterial homolog of Rh, has been determined. We aligned the sequence of Rhbg to that of AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, histidine 183 (H183), histidine 342 (H342), and tryptophan 230 (W230), within the hydrophobic pore where they presumably serve to control NH₃ transport. A fourth residue, phenylalanine 128 (F128) was positioned at the upper vestibule, presumably contributing to recruitment of NH₄⁺ We generated three mutations each of H183, H342, W230, and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane, whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH₃/NH₄⁺ and methyl amine (MA)/methyl ammonium (MA⁺)-induced currents, intracellular pH, and surface pH (pHs) among oocytes expressing the mutants, Rhbg, or injected with H₂O. In H183 and W230 mutants, NH₄⁺-induced current and intracellular acidification were inhibited compared with that of Rhbg, and MA-induced intracellular alkalinization was completely absent. Expression of H183A or W230A mutants inhibited NH₃/NH₄⁺- and MA/MA⁺-induced decrease in pHs to the level observed in H₂O-injected oocytes. Mutations of F128 did not significantly affect transport of NH₃ or NH₄⁺ These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter.

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