His‐183 is a Critical Structural Determinant of NH3/NH4+ Transport by Rhbg

Abstract

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 homologue of Rh, has been determined. We used bi‐dimensional hydrophobic cluster analysis to align the sequence of Rhbg to AmtB & identify important sites of Rhbg that may affect function. Our model positioned a conserved His‐183 within the hydrophobic pore where it presumably serves to control NH3 transport. Three mutations of His‐183 (to Ala, Leu or Asp) were generated and expressed in oocytes. To determine function, we measured NH3/NH4+ & methyl amine/ammonium (MA/MA+) ‐ induced currents and pHi in oocytes expressing the mutants and compared them to measurements in control oocytes or those expressing wild‐type Rhbg. In all mutants, except H183L, NH4+ current was smaller than that in oocytes expressing WT Rhbg. However MA+‐induced current was completely abolished in all mutants. In oocytes expressing H183L mutants, NH3/NH4+ caused significantly smaller changes in pHi & Vm than in Rhbg and MA/MA+ did not cause any change in pHi or Vm. The NH3/NH4+ and MA/MA+ ‐ induced pHi & Vm changes in the mutant were not different from those in H2O‐injected oocytes. These data indicate complete loss of function upon substituting His‐183. The loss of function indicates that this site is critical for transport by Rhbg Supported by VA Merit grant and by Tulane Institutional Fund