Electrogenic ammonium transport by renal Rhbg.
The recently cloned, non-erythrocyte Rh glycoproteins (Rhbg and Rhcg) are expressed in the intercalated cells of the renal collecting duct. The apical Rhcg and the basolateral Rhbg are likely involved in NH3 and/or NH4+ transport, yet the characteristics of this transport are not yet certain. In this study we investigated the mechanism of NH4+ transport by Rhbg and Rhcg expressed in Xenopus oocytes. We used a two-electrode voltage-clamp and ion-selective microelectrodes to measure NH4+-induced currents (I(NH4)) and changes in pHi, respectively. In oocytes expressing Rhcg, exposure to bath [NH4+] of 2.5-20 mM induced inward currents that were slightly more than those in H2O-injected (control) oocytes. I-V plots in the presence of NH4+ showed a small increase in slope conductance only at positive potentials. On the other hand, in oocytes expressing Rhbg, 5 mM NH4+ induced an inward I(NH4) of -79 nA, decreased pHi (DeltapHi) by 0.13 at a rate (dpHi/dt) of -2 7 x 10(-4) pH/s and depolarized the cell by 45 mV. These changes were significantly more than those in control oocytes. I-V plots in the presence of NH4+ showed substantial increase in conductance. Amiloride (1 mM) inhibited I(NH4), DeltapHi and dpHi/dt in oocytes expressing Rhbg but not in control oocytes. Raising bath [NH4+] in increments from 1 to 20 mM elicited a faster dpHi/dt, a larger decrease in pHi and a larger depolarization. Net NH4+ flux by Rhbg (estimated from dpHi/dt) was proportional to [NH4+] gradient and followed saturation kinetics with an apparent Km of 2.3 mM. Methyl ammonium (5 mM) induced a current of -63 nA in Rhbg oocytes but did not cause any change in control oocytes. These data indicate that: 1) Rhbg transport of NH4+ is electrogenic. 2) Methyl ammonium is transported by Rhbg. 3) NH4+ transport by Rhbg is saturated at high concentrations with Michaelis-Menten kinetics.
Department of Medicine, Section of Nephrology, SL-45, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, USA. nakhoul@tulane.edu
N L
NL
Nakhoul
E
E
Schmidt
S-M
SM
Abdulnour-Nakhoul
L L
LL
Hamm
D000584:Amiloride / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
D027682:Cation Transport Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008744:Methylamines; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; C486033:RhBG protein, mouse; C485880:Rhcg protein, mouse; D026941:Sodium Channel Blockers; D000641:Ammonia; D000584:Amiloride; C027451:methylamine