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1Department of Physiology, 5Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia; 2American Red Cross, Philadelphia, Pennsylvania; 3Division of Nephrology, University of Florida College of Medicine, Gainesville; and 4Nephrology and Hypertension Section, North Florida/South Georgia Veterans Health System, Gainesville, Florida
Submitted 8 April 2005 ; accepted in final form 24 August 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Rh proteins; Rh-associated proteins; ammonium
The nonerythroid homologs, designated RhBG and RhCG, are expressed in kidney, liver, brain, gastrointestinal tract, and skin (15, 22, 23, 39). In the kidney, RhBG and RhCG have been localized to the basolateral and apical membranes, respectively, in the collecting segment and collecting duct (33, 39), major sites of transepithelial ammonia/ammonium transport. In the intestinal tract, RhBG and RhCG are expressed in a similar polarized fashion in the duodenum and other regions of small and large intestine, other important sites for transepithelial ammonia transport (15). Recent studies in a mouse collecting duct cell line (mIMCD-3), which expresses basolateral RhBG and apical RhCG, revealed that both basolateral and apical ammonia/ammonium movement in these cells had characteristics of a protein-mediated transport process (13, 14). The transport activity was distinct from known potassium or sodium transporters, was independent of the membrane potential, and was stimulated by establishment of a transmembrane proton gradient, consistent with the behavior of erythrocyte RhAG expressed in heterologous cells (40). These studies suggested that the kidney RhBG and RhCG proteins may function in transepithelial movement of ammonia/ammonium.
In mammals, renal ammonium metabolism and transport are critical for whole animal acid-base balance. Ammonium ions are the primary component of net acid excretion under basal conditions and changes in renal ammonia metabolism and transport comprise the predominant renal response to metabolic acidosis (10, 21). Total ammonia secretion along the length of the collecting tubule has been previously thought to occur by passive NH3 diffusion. However, the recent observations concerning the function and localization of Rh glycoproteins suggest that ammonia secretion may possibly be mediated by members of the Rh family of proteins.
We have therefore undertaken experiments to characterize the possible ammonia/ammonium transport activity of the nonerythroid Rh glycoproteins, RhBG and RhCG. Each protein was expressed in X. laevis oocytes and functionally characterized utilizing two approaches: uptake of the ammonium analog [14C]methylamine (MA) and two-electrode voltage clamp (TEV) to assess the corresponding changes in membrane conductance, current, and reversal potential resulting from expression of the proteins in X. laevis oocytes.
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MATERIALS AND METHODS |
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Oocyte injection. Stage V and VI defolliculated oocytes obtained as described (16) were injected with 23–34 nl (1 µg/µl) of cRNA, or water for control, and placed in individual wells in 96-well plates containing 200 µl SOS containing (in mM) 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6, 200 mosM with 2.5 mM Na pyruvate and 100 µg/ml of gentamicin at 16°C. Oocytes were tested 3 days postinjection.
Western analysis. Recombinant protein expression in oocytes was analyzed as previously described (40). Western blots were probed with rabbit polyclonal anti-peptide antibodies to mouse RhBG and mouse RhCG (37, 39) and visualized with secondary horseradish peroxidase-conjugated, anti-rabbit IgG followed by ECL chemiluminescence (Amersham, Arlington Heights, IL).
MA flux assay. Radiolabeled [14C]methylammonium (CH3NH3+) (ICN, Irvine, CA) flux measurements were performed as described in detail previously (40). Briefly, six to eight oocytes were placed in 200 µl SOS uptake buffer containing 1 µCi/ml [14C]MA (20 µM) and various concentrations of unlabeled MA. For all experiments, radiotracer uptake was stopped by washing the oocytes six times with 1 ml of ice-cold unlabeled uptake buffer. Oocytes were then solubilized in 200 µl of 5% SDS and analyzed for radioactivity by liquid scintillation counting. In all experiments, water-injected control oocytes were evaluated in parallel, and uptake by controls was subtracted from that observed in test oocytes. Flux assay data were analyzed with Igor Pro software (Wavemetrics, Lake Oswego, OR). Initial uptake rates were determined from slopes of linear fits to uptake measured over 15 to 30 min, during which efflux was considered negligible. Apparent EC50 (Km) and Vmax values were derived by fitting experimental data with a simple Michaelis-Menten or Hill equation using Igor-Pro software with a nonlinear, least-squares fitting algorithm.
TEV experiments. TEV (16) was used to measure membrane currents, conductance, and voltage in cRNA-injected or water-injected control oocytes 3 days postinjection, while the oocytes were perfused with a solution containing (in mM) 96 NaCl, 2 KCl, 1 CaCl2, 1.8 MgCl2, 5 HEPES, pH 7.5, or with solutions containing (in mM) 0.5, 1, 5, or 10 NH4Cl or MACl, with correspondingly lower concentrations of NaCl to maintain the same osmolarity as detailed previously (40). The applied transmembrane potential (Vm) was ramped from –90 to +50 mV every 10 s. Transmembrane current (Im) and Vm were digitized at 1 kHz during the voltage ramps. In data analysis with Igor Pro software, a fifth-order polynomial was fitted to the raw, monotonically increasing Im-Vm data from each voltage ramp. The whole oocyte membrane conductance and the reversal potential (Vrev) were evaluated simultaneously as the slope (dIm/dVm) and the x-intercept of the polynomial, respectively.
[14C]MA flux assay under voltage clamp. Individual oocytes were voltage clamped to either –100 or to 0 mV as for conventional TEV experiments. In each experiment, an oocyte was allowed to equilibrate for 3–5 min in uptake buffer without MA, and then an equal volume of uptake buffer containing 2x MA (radioactive and unlabeled) was added. Oocyte membrane integrity was monitored visually and by whole oocyte membrane conductance measurements during the experiment. After the flux uptake period (15 min), cold flux buffer was perfused by flow through the bath to remove extracellular radioactivity. The electrodes were then removed, and the oocyte was analyzed for radioactive uptake, as above. Uptake of MA under voltage-clamp conditions was compared with flux uptake in oocytes that were impaled with TEV microelectrodes but were allowed to remain at resting potential (–30 to –50 mV) without voltage clamp.
Data analysis. The statistical significance of experimental results was evaluated by two-tailed t-test. P values <0.05 were considered significant. Data are plotted in Figs. 1–9 as arithmetic means ± SEM; n is reported as the number of different assays.
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RESULTS |
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RhBG and RhCG mediate uptake of MA.
To begin testing the hypothesis that RhBG and RhCG mediate movement of total ammonia and to characterize that transport, we expressed the nonerythroid Rh glycoproteins in X. laevis oocytes. cRNA for RhBG or RhCG was microinjected into oocytes and expression was monitored at 72 h after injection. Protein expression was detected after SDS-PAGE separation and immunoblotting with rabbit anti-peptide antibodies specific for RhBG or RhCG (39). RhBG migrated at 
52 kDa (Fig. 1A, inset), and RhCG at 58 kDa (Fig. 1B, inset), consistent with the reported molecular weights of these glycoproteins in mouse tissues (37, 39). The detected proteins were absent in water-injected control oocytes.
We previously showed that the radioactive analog tracer methylamine, [14C]CH3NH3+ (MA+/MA), which has been used to study ammonium transport in yeast and plants (20, 28, 32, 34), had the same relative affinity as NH3/NH4+ for the RhAG transport pathway (40). Therefore, we used the uptake of [14C]MA in cRNA-injected oocytes as a measure of NH3/NH4+ transport mediated by RhBG and RhCG. Expression of either mouse RhBG or mouse RhCG enhanced the rate of MA+/MA uptake compared with that seen in the water-injected controls by two- to threefold over controls at 500 µM substrate concentration (Fig. 1). Uptake was saturable at 
1 h (not shown), which is compatible with the uptake being a carrier-mediated process. In all subsequent flux experiments, water-injected control oocytes were evaluated, and the uptake reported was the uptake by the RhBG- or RhCG-expressing oocytes less the uptake by the control water-injected oocytes examined in parallel.
Effects of ammonium, organic ions, and inhibitors on MA transport.
To confirm that NH3/NH4+ is the actual substrate for RhBG and RhCG transport, competitive inhibition experiments were performed by measuring MA+/MA uptake in the presence of varying concentrations of NH4Cl. Uptake by RhBG and RhCG was significantly inhibited by ammonia (Fig. 2A). Differences in the concentration of ammonia required to inhibit RhBG- and RhCG-mediated MA+/MA uptake suggest that these proteins differ in their affinity for substrate. An inhibitory Hill equation fit to the data gave an IC50 for MA/MA+ transport inhibition by NH4Cl of 0.5 mM for RhBG and 2.9 mM for RhCG and a Hill coefficient of 1.4 for both.
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Transport kinetics. To determine the kinetics of transport, the rates of MA+/MA uptake were measured with increasing concentrations of substrate ranging from 20 µM to 50 mM. When fitted with a Hill equation, the Hill coefficient was 0.9 ±0.1, suggesting a lack of transporter cooperativity. Thus the data were well fitted with a Michaelis-Menten equation (Fig. 3, A and B). The EC50 (Km) for RhBG was 2.5 ± 0.5 mM, whereas the EC50 (Km) for RhCG was 10 ± 2 mM. These data suggest that RhBG and RhCG may function as low-affinity, high-capacity transporters and indicate that RhBG has a higher affinity for MA+/MA than RhCG.
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In water-injected oocytes not expressing RhBG or RhCG, high concentrations of extracellular NH4Cl (10 mM) increased membrane conductance and depolarized the oocytes (our unpublished observations and Ref. 2). This endogenous NH4Cl-dependent conductance complicates efforts to observe specific RhBG- or RhCG-mediated NH4Cl-dependent transport in oocytes in the presence of high (>5 mM) NH4Cl concentrations. The endogenous NH4Cl-dependent conductance is substantially smaller in the presence of the low NH4Cl concentrations (0.5–1 mM) at which RhBG and RhCG transport MA/MA+ effectively, as shown in the uptake studies (Fig. 3). Therefore, we looked for possible RhBG- or RhCG-mediated NH4Cl-dependent transmembrane conductances in the presence of 0.5 to 1 mM NH4Cl.
In a representative TEV experiment (Fig. 6), the transmembrane current Im in an RhBG-injected oocyte (dots) was monitored at various applied potentials, Vm, according to a voltage ramp protocol. From the fits (continuous curve) to the Im-Vm data, the whole cell current (Fig. 6B), the transmembrane conductance (Fig. 6C) at Vm = –50 mV, and the reversal potential (Fig. 6D) of the oocyte were evaluated simultaneously while the oocyte was continuously perfused with bath solutions containing various concentrations of NH4Cl.
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DISCUSSION |
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We found that both RhBG and RhCG mediated the transport of the ammonia/ammonium analog, MA, in a process that was competitively inhibited specifically by NH4Cl, but not by other amine compounds. Kinetic measurements revealed that RhBG had a higher affinity for MA/MA+ than RhCG. This observation is consistent with differences in the local NH3/NH4+ concentration in the collecting duct in the kidney where these proteins are localized (Fig. 9). RhCG is expressed on the apical cell surface facing the lumen (39) where the concentration of ammonium can reach 200 mM. In contrast, RhBG is localized to the basolateral surface facing the interstitium, where concentrations are significantly lower (<5 mM) (21). Thus the substrate affinities of RhBG and RhCG may be specific adaptations that enable these proteins to facilitate the vectorial transport of ammonia in the collecting duct in locations with disparate substrate concentrations.
Our data show that RhBG- and RhCG-mediated uptake of MA/MA+ was sensitive to the proton (H+) concentration in the extracellular buffer. Uptake was inhibited at acidic pH and enhanced at alkaline pH. Importantly, the enhanced uptake at alkaline pH was not due to increased concentration of unprotonated MA. In contrast, uptake rates in the presence of an equivalent concentration of unprotonated MA differed dramatically depending on the amount of protonated MA+ present, indicating that RhBG and RhCG respond to protonated substrate concentration in the extracellular medium. Nevertheless, the concentration of protonated MA+ was not the sole determinant of the rate of transport. Rather, the magnitude and direction of the H+ gradient also contribute to the rate of transport. Taken together, these data suggest a mechanism involving NH4+ and its movement across the plasma membrane by exchange with intracellular protons, resulting in electroneutral transport.
The membrane conductance monitored in TEV experiments was higher in the oocytes expressing RhBG and RhCG than in control ones, both in the absence as well as presence of extracellular NH4Cl. The nature of this conductance is not known, but our results suggest that it is unrelated to the Rh glycoprotein-mediated mechanism that transports MA. The TEV results revealed that oocytes have an endogenous NH4Cl-stimulated conductance even in the absence of RhBG/RhCG expression. The elevated conductance observed in RhBG/RhCG-expressing oocytes may reflect upregulation of this endogenous conductance.
Several groups have also reported enhanced conductance in RhBG (24, 30) or RhCG (1) cRNA-injected oocytes. Observed intracellular acidification, transmembrane currents, and cell depolarization in RhBG cRNA-injected oocytes exposed to 5 mM NH4Cl led Nakhoul et al. (30) to conclude that RhBG mediates electrogenic transport of protonated NH4+ across the plasma membrane. In contrast, Ludewig (24) observed a transient intracellular alkalization but also found enhanced inward currents in voltage-clamped, RhBG cRNA-injected oocytes perfused with 500 µM NH4Cl. Enhanced inward currents were also observed in voltage-clamped RhCG cRNA-injected oocytes (1). The results of these studies are reminiscent of the enhanced conductance observed in the experiments reported here. However, our direct measurements of substrate uptake in voltage-clamped oocytes now strongly indicate that the ammonia-stimulated conductance is distinct from the transport mechanism that mediates translocation of MA/MA+ by RhBG and RhCG. In agreement, Ludewig (24) also concluded that inward currents observed were not directly associated with the mechanism that mediates RhBG transport. Of note, many studies have observed the upregulation of endogenous oocyte conductances in response to recombinant protein expression (3, 5, 31, 35, 36). Because TEV conductance and current measurements in oocytes can be confounded by movement of ions other than those under study, we used radioactive labeled MA to directly monitor substrate movement across the membrane in RhBG- and RhCG-expressing oocytes under voltage-clamp conditions. Importantly, MA uptake was unaffected by large changes in the potential difference across the plasma membrane. These results strongly suggest that Rh-associated glycoprotein-mediated ammonia uptake is intrinsically an electroneutral transport process. Taken together with the evidence that the transport process involves NH4+ and an oppositely directed H+ gradient, we conclude that both RhBG and RhCG expressed in X. laevis oocytes mediate electroneutral transport of NH4+ and H+, which results in the net transport of NH3 across the plasma membrane of the cell.
Members of the Amt/MEP/Rh family of proteins, to which RhBG and RhCG belong, are expressed in a broad range of organisms, from bacteria and yeast to plants and mammals. The structure of E. coli AmtB has recently been solved by two groups using X-ray crystallography (17, 42). The structure reveals that the bacterial protein transports uncharged NH3 via a unique mechanism. AmtB contains an extracellular vestibule that recruits NH4+ cations, but the hydrophobic pore through the membrane is narrow. The NH4+ is stripped of a proton in the vestibule, enabling uncharged NH3 to pass through the pore via weak interactions with hydrogen bond donors that line the pore. A proton is released back to the extracellular aqueous phase during the process. Conversely, NH3 is reprotonated at the intracellular face of the channel. The net result is NH4+ transport in exchange for H+. The mechanisms regarding NH3/NH4+ transport by AmtB inferred from analyses of the crystal structures are entirely consistent with our experimental data showing that the mammalian Rh-associated glycoproteins (RhAG, RhBG, and RhCG) mediate electroneutral transport with net transfer of NH3. The extracellular alkaline pH stimulation and transmembrane proton gradient sensitivity seen with the Rh-associated glycoproteins are consistent with the requirement for transfer of a proton to the extracellular aqueous phase concurrent with movement of NH3 across the membrane. The structures support our original hypothesis that the transport mechanism involves exchange of NH4+ for H+ (40), although the molecular details are distinct from those of a NH4+/H+ exchanger mechanism.
Nevertheless, the relevance of the AmtB structure for the mechanism of NH3 transport by Rh-associated glycoproteins remains to be more fully established. Notably, in contrast to the mammalian Rh-associated glycoproteins, pH has not been observed to affect uptake by the bacterial (29), plant (25), or yeast (27) proteins. Thus the mechanism of transport by the mammalian proteins may differ from the plant, bacterial, and yeast transporters. It has been proposed that the bacterial and plant transporters require a membrane potential gradient (28, 29, 32). However, because this conclusion was based on the use of protonophores to dissipate membrane potential, this interpretation may have been confounded by the fact that the membrane potential in these organisms is maintained by proton pumps. Thus an alternative explanation for those observations is that protonophores interfered with uptake because they eliminated the proton gradient required for uptake.
In the kidney, RhBG and RhCG may function to mediate transepithelial transfer of NH3/NH4+ from the interstitium to the lumen in the collecting duct. In support, mIMCD grown on permeable support membranes demonstrate both apical and basolateral plasma membrane ammonia-inhibitable [14C]MA uptake, compatible with a transporter-mediated apical and basolateral membrane process (13, 14). The transport kinetics measured in the mIMCD cells were very similar to those obtained in the oocyte experiments reported here. Figure 9 is a diagrammatic representation of the medullary collecting duct showing the location of RhBG and RhCG and indicating the approximate local concentration of ammonia (both NH4+ and NH3) and the pH. The basolateral pH may vary along the collecting duct, progressing from slightly more acidic than systemic pH to slightly more alkaline (7, 8, 11). Extracellular ammonia concentrations also vary along the length of the collecting duct (reviewed in Ref. 6) and the collecting duct luminal NH4+ concentration can exceed 200 mM. However, our study indicates that RhBG and RhCG transport net NH3 across the plasma membrane through an electroneutral process involving NH4+/H+ exchange. This movement of net NH3 is determined by the transmembrane concentration gradients of NH4+ and H+. The net transport of NH3 mediated by RhBG and RhCG from the interstitium through the epithelial cell cytoplasm to the collecting duct lumen proceeds down the NH3 concentration gradient (from 50–90 through 50 to 7–35 µM) and therefore requires no energy input. The rates of RhBG- and RhCG-mediated transport are determined by the pH and NH4+ concentrations of the interstitium, epithelial cell cytoplasm, and collecting duct lumen, which are regulated by various cellular mechanisms. Indeed, transepithelial ammonia secretion rate in the collecting duct is known to be determined by both the ammonia concentration and the pH gradient (9, 12), and increasing luminal H+ concentration stimulates transepithelial ammonia secretion.
In mammals, NH3/NH4+ secretion is critical for acid-base balance. The localization of RhBG and RhCG to the connecting segment and collecting duct, the final sites of renal acid secretion, positions them to affect acid-base regulation. The causes of some forms of distal renal tubular acidosis, an inherited disorder affecting acid-base transport in the renal collecting duct, have not yet been uncovered, making RhCG and RhBG prime targets for investigation. Functional studies of the kidney and erythrocyte Rh-associated proteins promise to contribute to understanding the role of these proteins in ammonia/ammonium elimination.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-subunit in Xenopus oocytes induces an endogenous voltage-gated large diameter pore. J Physiol 535: 407–417, 2001.This article has been cited by other articles:
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 H.-W. Lee, J. W. Verlander, J. M. Bishop, P. Igarashi, M. E. Handlogten, and I. D. Weiner Collecting duct-specific Rh C glycoprotein deletion alters basal and acidosis-stimulated renal ammonia excretion Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1364 - F1375. [Abstract] [Full Text] [PDF]
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A. C. N. Brown, D. Hallouane, W. J. Mawby, F. E. Karet, M. A. Saleem, A. J. Howie, and A. M. Toye RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1279 - F1290. [Abstract] [Full Text] [PDF]
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Represent experimental data, and the curve represents the Michaelis-Menten fit with the tabulated parameters. The EC50 (Km) for RhBG was 2.5 mM compared with an EC50 (Km) for RhCG of 
