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1 Department of Medicine, Section of Nephrology, and Department of Physiology, Tulane University School of Medicined, New Orleans, LA, USA
2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: nakhoul{at}tulane.edu.
Rhbg is one of two recently cloned non-erythroid glycoproteins belonging to the Rh antigen family. Rhbg is expressed in basolateral membranes of intercalated cells of the kidney cortical collecting duct and some other cell types of the distal nephron and may function as NH4+ transporters. The aim of this study was to characterize the role of Rhbg in transporting NH4+. To do so we expressed Rhbg in Xenopus oocytes. Two-electrodes voltage clamp and H+-selective microlectrodes were used to measure NH4+ currents, I-V plots and intracellular pH (pHi). In oocytes expressing Rhbg, 5 mM NH4+ induced an inward current of 93±7.7 nA (n=20) that was significantly larger than that in control oocytes of -29±7.1 nA (p<0.005). Whole cell conductance, at all tested potentials (-60 to +60 mV) was significantly more in oocytes expressing Rhbg as compared to H2O-injected oocytes. In Rhbg oocytes, 5 mM NH4+ depolarized the oocyte by 28±3.6 mV and decreased pHi by 0.30±0.04 at a rate of -20±2.5 x 10-4 pH/sec. In control oocytes, 5 mM NH4+ depolarized Vm by only 20±5.8 mV and pHi decreased by 0.07±0.01 at a rate of -2.7±0.6 x10-4 pH/sec. Raising bath [NH4+] in increments from 1 to 20 mM elicited a proportionally larger decrease in pHi (
pHi), larger depolarization ( Vm) and a faster rate of pHi decrease. Bathing Rhbg oocytes in 20 mM NH4+ induced an inward current of 140±7 nA that was not significantly different from 178±23 nA induced in H2O-injected (control) oocytes. The rate of pHi decrease induced by increasing external [NH4+] was significantly faster in Rhbg than in H2O-injected oocytes at all external NH4+ concentrations. In oocytes expressing Rhbg, net NH4+ influx (estimated from NH4+-induced H+ influx) as a function of external [NH4+] saturated at higher [NH4+] with a Vmax of ~30.8 and an apparent Km of 2.3 mM (R2=0.99). These data strongly suggest that Rhbg is a specific electrogenic transporter of NH4+.
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