pH sensitivity of ammonium transport by Rhbg

Rhbg is a membrane glycoprotein that is involved in NH3/NH4+ transport. Several models have been proposed to describe Rhbg, including an electroneutral NH4+/H+ exchanger, a uniporter, an NH4+ channel, or even a gas channel. In this study, we characterized the pH sensitivity of Rhbg expressed in Xenopus oocytes. We used two-electrode voltage clamp and ion-selective microelectrodes to measure NH4+-induced [and methyl ammonium (MA+)] currents and changes in intracellular pH (pHi), respectively. In oocytes expressing Rhbg, 5 mM NH4Cl (NH3/NH4+) at extracellular pH (pHo) of 7.5 induced an inward current, decreased pHi, and depolarized the cell. Raising pHo to 8.2 significantly enhanced the NH4+-induced current and pHi changes, whereas decreasing bath pH to 6.5 inhibited these changes. Lowering pHi (decreased by butyrate) also inhibited the NH4+-induced current and pHi decrease. In oocytes expressing Rhbg, 5 mM methyl amine hydrochloride (MA/MA+), often used as an NH4Cl substitute, induced an inward current, a pHi increase (not a decrease), and depolarization of the cell. Exposing the oocyte to MA/MA+ at alkaline bath pH (8.2) enhanced the MA+-induced current, whereas lowering bath pH to 6.5 inhibited the MA+ current completely. Exposing the oocyte to MA/MA+ at low pHi abolished the MA+-induced current and depolarization; however, pHi still increased. These data indicate that 1) transport of NH4+ and MA/MA+ by Rhbg is pH sensitive; 2) electrogenic NH4+ and MA+ transport are stimulated by alkaline pHo but inhibited by acidic pHi or pHo; and 3) electroneutral transport of MA by Rhbg is likely but is less sensitive to pH changes

Substrate specificity of Rhbg: ammonium and methyl ammonium transport

Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pHi) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pHi consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pHi. MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pHi increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pHi in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg