pH sensitivity of ammonium transport by Rhbg

Abstract

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 (pH_i), respectively. In oocytes expressing Rhbg, 5 mM NH₄Cl (NH₃/NH₄⁺) at extracellular pH (pH_o) of 7.5 induced an inward current, decreased pH_i, and depolarized the cell. Raising pH_o to 8.2 significantly enhanced the NH₄⁺-induced current and pH_i changes, whereas decreasing bath pH to 6.5 inhibited these changes. Lowering pH_i (decreased by butyrate) also inhibited the NH₄⁺-induced current and pH_i decrease. In oocytes expressing Rhbg, 5 mM methyl amine hydrochloride (MA/MA⁺), often used as an NH₄Cl substitute, induced an inward current, a pH_i 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 pH_i abolished the MA⁺-induced current and depolarization; however, pH_i 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 pH_o but inhibited by acidic pH_i or pH_o; and 3) electroneutral transport of MA by Rhbg is likely but is less sensitive to pH changes.

several studies have shown that Rhbg and Rhcg are involved in transport of NH₃/NH₄⁺ (1, 11, 12, 14–16, 20, 24, 27, 28). In our previous studies (19, 22), we demonstrated that electrogenic NH₄⁺ transport is enhanced in oocytes expressing Rhbg. We have also shown that methyl amine (MA) and methyl ammonium (MA⁺) are transported by Rhbg, yet their mode of transport differs from that of NH₃/NH₄⁺ (22). Whereas transport of NH₃/NH₄⁺ by Rhbg is predominantly electrogenic, transport of MA/MA⁺ has an apparent electroneutral as well as an electrogenic component. We have also demonstrated that amiloride is a partial inhibitor of NH₃/NH₄⁺ and MA/MA⁺ transport by Rhbg. Other functional characteristics of Rhbg, such as the effect of pH, are yet to be determined.

The physiological roles of Rhbg and Rhcg in acid-base transport and in renal NH₃/NH₄⁺ handling are becoming more evident. Rhcg is highly expressed in the epididymis, where it may be involved in the mechanisms of acidifying the epididymal fluid needed for maturation of sperm (3). Interestingly, Rhcg knockout mice were found to have lower pH of the epididymal fluid compared with wild type. In the kidney, chronic metabolic acidosis increased Rhcg protein expression in cells of the medullary collecting duct (CD) (25). The same study indicated an increase in apical and basolateral Rhcg expression. Metabolic studies demonstrated that mice lacking Rhcg had impaired urinary NH₄⁺ excretion in response to acid loads (3). A recent study on mice with renal CD-specific Rhcg deletion (CD-KO) (9) showed that urinary NH₄⁺ excretion was less in KO mice than in control mice and that after acid loading CD-KO mice developed more severe metabolic acidosis than control mice. Studies on Rhbg showed that genetic deletion of pendrin, an apical Cl⁻/HCO₃⁻ exchanger in intercalated cells, decreased Rhbg expression. Recently, Bishop et al. (2) generated intercalated cell-specific Rhbg KO mice and demonstrated that when acid loaded urinary ammonium excretion was significantly less in KO mice versus control mice.

The pH sensitivity of Rhbg is of significant importance for several reasons. First, a change in pH will shift the equilibrium of the ammonia-to-ammonium balance with acidic pH favoring increased [NH₄⁺]/[NH₃] and thus may affect transport of NH₄⁺ by Rhbg. Second, it has been proposed that Rhbg may function as an NH₄⁺/H⁺ exchanger that is driven by the H⁺ gradient (12, 14, 27). Therefore, a pH change will also affect transport of NH₄⁺ by Rhbg. Third, a transport model based on the crystal structure of AmtB (a bacterial homolog of Rh proteins) suggests a mechanism of recruitment of extracellular NH₄⁺ and eventual transport and release of neutral NH₃ intracellularly. Both processes are likely pH sensitive. Finally, pH sensitivity of this transporter is probably highly significant in acid-base disturbances where renal Rhbg, for example, may play an important role in the adaptive response to acidosis. For these reasons we investigated the sensitivity of Rhbg to pH. To do so, we examined the effects of pH changes on NH₃/NH₄⁺ and MA/MA⁺ transport by Rhbg.

METHODS

All experiments were conducted on oocytes expressing Rhbg or injected with H₂O as a control. NH₃/NH₄⁺ and MA/MA⁺ transport were assessed from measurements of changes in intracellular pH (pH_i), membrane potential (V_m), and whole cell currents induced by exposing the oocytes to bath solutions containing 5 mM NH₄Cl or 5 mM methyl ammonium chloride. Bath solutions were HCO₃⁻ free and buffered with HEPES. The standard bathing solution was amphibian ND96 medium and had the following composition in mM: 100 NaCl, 2 KCl, 1.2 CaCl₂, and 5 HEPES buffered to pH 7.5. The NH₃/NH₄⁺ solutions had 5 mM NH₄Cl replacing NaCl. Similarly, the MA/MA⁺ solution had 5 mM methyl ammonium chloride replacing NaCl. The standard bath solution had a pH of 7.5. Other bath solutions used had a pH of either 8.2 or 6.5. These solutions were prepared by titrating the specified solution with NaOH or HCl, respectively. The osmolality of every solution was ∼200 mosmol/kgH₂O.

Isolation of Oocytes

All experiments were conducted on oocytes in stage 5 or 6. Isolation of oocytes from Xenopus laevis frogs (Xenopus Express) was done according to standard operating procedures as described previously (19, 21). Briefly, the frog was anesthetized in water containing 0.2% tricaine adjusted to pH 7.2 with NaDH and buffered with 5 mM HEPES. The oocytes were extracted from a 1-cm incision in the abdominal wall where a lobe of the ovary was externalized and its distal portion cut. The oocytes were isolated by treating the excised piece of ovary with sterile-filtered Ca²⁺-free solution containing collagenase type I-A (2–3 mg/ml) for 30–40 min, followed by several washes in Ca²⁺-free solution. Free oocytes were rinsed several times with sterile OR3 medium, sorted, and then stored at 18°C. The protocols describing animal handling procedures and isolation of oocytes were approved by the Institutional Animal Care and Use Committee of Tulane University Medical School.

Capped RNA Preparation and Injection of Oocytes

Capped RNA (cRNA) for Rhbg was prepared from transcribed Rhbg cDNA with the mMessage mMachine T7 transcription kit from Ambion (Foster City, CA) as described previously (22). The concentration of cRNA was determined by UV absorbance, and its quality was assessed by formaldehyde/MOPS/1% agarose gel electrophoresis. Oocytes in OR3 medium were visualized with a dissecting microscope and injected with 50 nl of cRNA for Rhbg (0.2 μg/μl, for a total of 10 ng of RNA). Control oocytes were injected with 50 nl of sterile H₂O. The sterile pipettes had tip diameters of 20–30 μm. They were backfilled with paraffin oil and were connected to a Nanoject motor-driven pipette (Drummond Scientific). Injected oocytes were used on the third day after injection with cRNA and for the following 4–5 days.

pH and Electrophysiological Measurements in Frog Oocytes

pH_i measurements and voltage-clamp experiments were conducted as previously reported (19, 22) and are briefly described here. pH_i was measured by H⁺-selective microelectrodes of the liquid ion exchanger type (H⁺ ionophore 1, cocktail B) obtained from Fluka (Buchs, Switzerland). The microelectrodes were manufactured as described by Siebens and Boron (26). The electrodes were fitted with a holder with an Ag-AgCl wire attached to a high-impedance probe of a WPI FD-223 electrometer and calibrated. The pH electrodes were calibrated in standard solutions of pH 6 and 8. All the electrodes used in this study had a slope of >56 mV/pH. The pH electrodes were not selective to MA⁺ over a concentration range from 1 to 20 mM.

Initial rates of change of pH_i (dpH_i/dt) were determined by fitting pH_i vs. time to a linear regression line. Statistical significance was judged from Student's t-tests. Whenever possible, measurements were determined under control and test conditions in the same oocyte.

Two-Electrode Voltage Clamp

Two-electrode voltage clamp (OC-725, Warner Instruments, Hamden, CT) was used to measure whole cell currents. Electrodes were filled with 3 M KCl solution and had resistances of 1–4 MΩ. Bath electrodes were also filled with 3 M KCl and were directly immersed in the chamber. Oocytes were clamped at −60 mV, and long-term readings of current were sampled at a rate of once per second. Inward flow of cations is defined by convention as inward current (negative current).

Abbreviations

NH₃/NH₄⁺ refers to an equilibrated mix of ammonia gas (NH₃) and ammonium ions (NH₄⁺) obtained by dissolving NH₄Cl in solution. MA/MA⁺ refers to methyl amine (MA) in equilibrium with methyl ammonium (MA⁺) obtained by dissolving methyl amine hydrochloride salt in solution. pH_i refers to intracellular pH; pH_o is extracellular or bath pH.

RESULTS

Transport of NH₃/NH₄⁺ by Rhbg

Basic effect.

Several studies have shown that Rhbg and Rhcg are involved in transport of NH₃/NH₄⁺. In our previous studies, we demonstrated that electrogenic NH₄⁺ transport is enhanced in oocytes expressing Rhbg. Thus in oocytes expressing Rhbg, exposure to NH₃/NH₄⁺ caused a fast and significant decrease in pH_i, a depolarization of the cell, and an inward current (19, 22). Characteristically absent was any pH_i increase, suggesting minimal activation of NH₃ transport (relative to NH₄⁺) that would normally cause alkalinization. These changes were significantly more pronounced than NH₃/NH₄⁺-induced changes in H₂O-injected oocytes. However, because H₂O-injected oocytes exhibited similar, albeit much smaller, changes induced by NH₃/NH₄⁺, it was suggested that there is endogenous background NH₄⁺ transport in native oocytes (4–6, 19, 21).

On the other hand, we showed that MA/MA⁺, often used to substitute for NH₃/NH₄⁺, is also a substrate of Rhbg. However, exposure to MA/MA⁺ caused an increase in pH_i (not a decrease), a depolarization of the cell, and an inward current (19, 22). In H₂O-injected oocytes there was no significant MA/MA⁺ transport that can be detected by similar measurements. This suggests that transport of MA/MA⁺ in H₂O-injected oocytes is much less than in oocytes expressing Rhbg. In this study, we relied on measurements of pH_i, V_m, and voltage-clamped current in oocytes expressing Rhbg to characterize the pH sensitivity of this transporter.

pH Dependence of NH₄⁺ Transport by Rhbg

Sensitivity to extracellular pH.

EFFECT OF EXTRACELLULAR ALKALINIZATION ON NH₄⁺-INDUCED CURRENT.

To assess the role of pH_o, we measured the NH₄⁺-induced current (voltage clamped at −60 mV) at normal bath pH of 7.5 and compared it to that at alkaline pH of 8.2. In oocytes expressing Rhbg (Fig. 1A), exposing the cell to 5 mM NH₄Cl at bath pH of 7.5 caused an inward current (segment ab) that was readily reversed upon removal of bath NH₄⁺ (segment bc). At point c, bath pH was raised to 8.2 with no discernable effect on current (segment cd). Exposing the same oocyte to 5 mM NH₄Cl at a bath pH of 8.2 caused a larger inward current (segment de) compared with that at pH 7.5. The NH₄⁺-induced current was reversed upon removal of bath NH₄⁺ (segment ef). At point f, bath pH was returned to normal (7.5). Another NH₄⁺ pulse at pH 7.5 was repeated, causing an inward current (segment gh) that was smaller than that at pH 8.2. In nine paired experiments, the NH₄⁺-induced current averaged −67 ± 6.2 nA at pH_o 7.5 compared with −116 ± 12.8 nA at pH_o 8.2 (P < 0.001). These experiments suggested that raising pH_o in oocytes expressing Rhbg enhanced electrogenic NH₄⁺ transport.

Fig. 1.

Effect of alkaline extracellular pH (pH_o) on NH₄⁺-induced current (I). A: in oocytes expressing Rhbg, exposure to NH₃/NH₄⁺ (5 mM) at pH 7.5 induced an inward current (clamped at −60 mV) of −67 ± 6.2 nA (segment ab). Exposing the oocyte to 5 mM NH₃/NH₄⁺ at pH_o of 8.2 induced a significantly larger inward current of −116 ± 12.8 nA (segment de). A change in bath pH from 7.5 to 8.2 (or back to 7.5) by itself did not cause any change in current (segments cd and fg, respectively). Segment ghi is a repeat of the 1st control pulse (NH₃/NH₄⁺ at pH_o of 7.5), causing similar results. B: in H₂O-injected oocytes, exposure to NH₃/NH₄⁺⁺ at bath pH of 7.5 induced an inward current (segment ab) that was significantly enhanced when the oocyte was exposed to NH₃/NH₄⁺ at pH 8.2 (segment de). Segment ghi is a repeat of the initial NH₃/NH₄⁺ pulse at pH 7.5, with comparable results. C: summary graph indicating inward currents induced by 5 mM NH₃/NH₄⁺ at bath pH of 7.5 and 8.2 in oocytes expressing Rhbg and those injected with H₂O. NH₄⁺-induced current was enhanced by alkaline pH in Rhbg and control oocytes. *Statistical significance between pH 7.5 and 8.2. Std HEPES, standard HEPES.

We then investigated whether extracellular alkalinization affected NH₄⁺ transport in H₂O-injected oocytes. As shown in Fig. 1B, exposing H₂O-injected (control) oocytes to 5 mM NH₄Cl at pH_o 7.5 caused an inward current (segment ab), which was readily reversed upon removal of bath NH₄Cl (segment bc). By comparison to Rhbg, the NH₄⁺-induced current in H₂O-injected oocyte was significantly smaller than in Rhbg oocytes (compare with Fig. 1A). At point c, the pH in the bath was raised to 8.2, causing no change in current (segment cd). Addition of 5 mM NH₄Cl at pH_o 8.2 caused a significantly bigger inward current (segment de), which readily reversed upon removal of bath NH₄⁺ (segment ef). The rest of the experiment was a repeat of the first NH₄⁺ pulse. Bath pH was returned to normal 7.5 with no change in current (segment fg). The oocyte was then reexposed to 5 mM NH₄Cl at pH 7.5, causing an inward current (segment gh) that readily recovered upon removal of bath NH₄⁺ (segment hi). This second pulse at pH_o 7.5 (segment ghi) was comparable to the first NH₄⁺ pulse (segment abc) and much smaller than the NH₄⁺ pulse at higher bath pH (segment cde). In six paired experiments on H₂O-injected oocytes, the NH₄⁺-induced current averaged −22 ± 3.1 nA at pH_o 7.5 but was increased to −142 ± 6.8 nA (P < 0.001) at pH_o 8.2.

The experiments of Fig. 1 clearly indicate that electrogenic NH₄⁺ transport is enhanced at alkaline pH. This effect is evident in both H₂O-injected oocytes and those expressing Rhbg. These results are summarized in Fig. 1C. It is important to note that whereas raising pH_o activated NH₄⁺ transport in oocytes in general, this does not exclude the possibility that alkaline pH may have activated transport by Rhbg also.

EFFECT OF EXTRACELLULAR ALKALINIZATION ON NH₄⁺-INDUCED PH_I CHANGES.

In the next set of experiments, we investigated the effect of raising pH_o on the NH₄⁺-induced pH_i changes. In oocytes expressing Rhbg (Fig. 2), 5 mM NH₄Cl in the bath caused a pH_i decrease and depolarization of the cell (segment ab) as demonstrated in earlier studies. Removal of bath NH₄Cl reversed these changes, and pH_i and V_m fully recovered (segment bc). Switching bath pH to 8.2 did not cause significant pH_i or V_m changes (segment cd). Adding 5 mM NH₄Cl to the bath at pH 8.2 caused a faster decrease in pH_i (segment de) and depolarized the cell. These changes were reversed upon removal of bath NH₄⁺ (segment ef).

Fig. 2.

Effect of alkaline bath pH on NH₃/NH₄⁺-induced intracellular pH (pH_i) and membrane potential (V_m) changes in oocytes expressing Rhbg. Exposing oocytes to 5 mM NH₃/NH₄⁺ at pH 7.5 caused a decrease in pH_i and depolarization of the cell (segment ab). Removal of NH₃/NH₄⁺ reversed these changes (segment bc). Raising bath pH from 7.5 to 8.2 did not cause any significant pH_i or V_m changes (segment cd). Exposing the oocyte to NH₃/NH₄⁺ at bath pH of 8.2 caused a decrease in pH_i and depolarization (segment de) that readily reversed when NH₃/NH₄⁺ was removed from the bath (segment ef). The rate of NH₃/NH₄⁺-induced pH_i decrease at pH_o 7.5 was significantly slower than at pH_o of 8.2. Numbers next to the pH_i tracing indicate the rate of pH_i decrease in 10⁻⁴ pH units/s.

Figure 3 summarizes these results on 13 paired experiments on oocytes expressing Rhbg. As indicated, bath NH₃/NH₄⁺ at pH 7.5 caused pH_i to decrease by 0.09 ± 0.01 pH units at a rate of −21 ± 3.1 × 10⁻⁴ pH units/s and depolarized the cell by 42 ± 3.1 mV. On the other hand, at pH_o 8.2 NH₄⁺ caused pH_i to decrease by 0.11 ± 0.01 pH units (P < 0.01) at a rate of −30 ± 2.8 × 10⁻⁴ pH units/s (P < 0.01). However, the V_m change of 45 ± 2.1 mV at pH 8.2 was not significantly different from the NH₄⁺-induced depolarization of 42 ± 3.1 mV at pH 7.5 (P > 0.05).

Fig. 3.

Summary of effect of extracellular alkalinization on NH₃/NH₄⁺-induced changes in oocytes expressing Rhbg. *Statistical significance (P < 0.05). dpH_i/dt, rate of change in pH_i.

Similar experiments were repeated on H₂O-injected oocytes. As shown in Fig. 4, exposing control oocytes to 5 mM NH₄Cl caused a pH_i decrease of 0.02 pH units at a rate of −4.0 × 10⁻⁴ pH units/s (segment ab). V_m depolarized by 45 mV. These effects were fully reversed upon removal of bath NH₄Cl (segment bc). At point c, bath pH was raised to 8.2, with no significant effects on pH_i or V_m (segment cd). Exposing the cell to 5 mM NH₄Cl at pH 8.2 caused a pH_i decrease of 0.06 pH units at a rate of −9.3 × 10⁻⁴ pH units/s and a depolarization of 54 mV (segment de). These changes fully recovered when NH₄⁺ was removed from the bath (segment ef). In six paired experiments, bath NH₄Cl at pH 7.5 induced an average decrease in pH_i of 0.04 ± 0.01 pH units at a rate of −6 ± 3.0 × 10⁻⁴ pH units/s and V_m depolarized by 35 ± 2.6 mV. Bath NH₄Cl at pH_o 8.2 caused pH_i to decrease by 0.07 ± 0.02 pH units at a rate of −12 ± 2.2 × 10⁻⁴ pH units/s, and V_m depolarized by 52 ± 3.2 mV. The rates of pH_i decrease and depolarization were significantly different from those induced by NH₄⁺ at pH 7.5 (P < 0.01). In contrast to the NH₄⁺-induced current, the pH_i and voltage changes were clearly larger in Rhbg-expressing oocytes than in H₂O-injected oocytes.

Fig. 4.

Effect of extracellular alkalinization on NH₃/NH₄⁺-induced pH_i and V_m changes in H₂O-injected oocytes. Exposing oocytes to 5 mM NH₃/NH₄⁺ at bath pH of 7.5 caused a slow decrease in pH_i (segment ab) and significant depolarization of the cell. Removal of bath NH₃/NH₄⁺ reversed these changes (segment bc). Raising bath pH to 8.2 did not cause any effect on pH_i or V_m (segment cd). Exposing the oocyte to 5 mM NH₃/NH₄⁺ at bath pH of 8.2 slightly but significantly enhanced the rate of NH₄⁺-induced pH_i decrease (segment de). pH_i and V_m readily recovered when NH₃/NH₄⁺ was removed from the bath.

EFFECT OF EXTRACELLULAR ACIDIFICATION ON NH₄⁺-INDUCED CURRENT.

In the next set of experiments, we examined the effect of lowering bath pH on NH₄⁺-induced current in Rhbg and H₂O-injected oocytes. In oocytes expressing Rhbg (Fig. 5A), 5 mM NH₄Cl in the bath at pH 7.5 induced an inward current (segment ab) as previously observed. Removal of NH₄Cl reversed this effect fully (segment bc). At point c, bath pH_o was decreased to 6.5 with no effect on current (segment cd). However, switching the bath solution to a pH of 6.5 completely inhibited the NH₄⁺-induced current usually observed at pH 7.5 (segment de). At point e, the bath solution was returned to standard HEPES (pH 7.5) with no effect in current.

Fig. 5.

Effect of extracellular acidification on NH₄⁺-induced current. A: in oocytes expressing Rhbg, exposure to 5 mM NH₃/NH₄⁺ at bath pH of 7.5 caused an inward current (segment ab) that readily reversed on removal of bath NH₃/NH₄⁺. Lowering bath pH to 6.5 did not cause any change in current (segment cd). Exposing the oocyte to 5 mM NH₃/NH₄⁺ at bath pH of 6.5 completely inhibited the NH₄⁺- induced change in current (segment de). B: in H₂O-injected oocytes, similar effects of bath pH were observed: 5 mM NH₃/NH₄⁺ at pH_o 7.5 caused an inward current (segment ab); 5 mM NH₃/NH₄⁺ in the bath at pH_o 6.5 did not induce any change in current (segment de).

Similar experiments were conducted on H₂O-injected oocytes. As shown in Fig. 5B, exposing control oocytes to 5 mM NH₄Cl at pH 7.5 caused an inward current (segment ab), which readily recovered upon removal of bath NH₄⁺ (segment bc). As previously reported, the NH₄⁺-induced current in H₂O-injected oocytes was significantly less than that caused by NH₄⁺ in oocytes expressing Rhbg (compare segment abc in Fig. 5, A and B). At point c, bath pH was decreased to 6.5, causing no change in the whole cell current (segment cd). Exposing the oocyte to 5 mM NH₄Cl at pH 6.5 did not cause any significant change in current as well (segment de). At point e, the bath solution was switched back to standard HEPES with no effect on current (segment ef).

In 11 experiments on oocytes expressing Rhbg, NH₄⁺-induced inward current at pH 7.5 averaged −56 ± 4.7 nA and was completely blocked at pH 6.5. In seven experiments on H₂O-injected oocytes, NH₄⁺-induced inward current at pH 7.5 averaged −31 ± 6.4 nA and was also completely blocked at pH 6.5. As stated above, the NH₄⁺ current at pH 7.5 in oocytes expressing Rhbg was significantly larger than that in H₂O-injected oocytes (P < 0.01). The results of these experiments indicate that electrogenic NH₄⁺ transport in control oocytes and those expressing Rhbg are inhibited at low pH_o.

EFFECT OF EXTRACELLULAR ACIDIFICATION ON NH₄⁺-INDUCED PH_I CHANGES.

To further investigate the role of pH_o, we measured the NH₃/NH₄⁺-induced pH_i changes at a low bath pH of 6.5. As shown in Fig. 6, exposing an Rhbg-expressing oocyte to 5 mM NH₄Cl in the bath caused the usual pH_i decrease (segment ab) and depolarization of the cell. Removal of bath NH₄Cl reversed these changes completely (segment bc). Switching bath pH to 6.5 caused a very small decrease in pH_i and V_m (segment cd). Exposing the oocyte again to 5 mM NH₄Cl but at pH_o of 6.5 caused a smaller decrease in pH_i (segment de) at a slower rate of acidification (−15 vs. −39 × 10⁻⁴ pH units/s). Moreover, the NH₄⁺-induced depolarization was significantly blunted. Removal of bath NH₄Cl fully reversed these effects, and pH_i and V_m recovered (segment ef). In six paired experiments, 5 mM NH₃/NH₄⁺ at bath pH of 7.5 decreased pH_i by 0.14 ± 0.001 pH units at a rate of −29 ± 4.9 × 10⁻⁴ pH units/s and V_m depolarized by 35 ± 3.6 mV. At bath pH of 6.5, 5 mM NH₃/NH₄⁺ decreased pH_i by 0.06 ± 0.03 pH units at a rate of −13 ± 1.4 × 10⁻⁴ pH units/s and V_m depolarized by only 2.0 ± 1.8 mV.

Fig. 6.

Effect of extracellular acidification on NH₃/NH₄⁺-induced pH_i and V_m changes in oocytes expressing Rhbg. Exposing the oocyte to 5 mM NH₄Cl at pH 7.5 caused a decrease in pH_i and depolarization (segment ab) that readily reversed upon removal of NH₄Cl from the bath (segment bc). Lowering bath pH to 6.5 did not cause any significant change in pH_i but depolarized the cell slightly (segment cd). Exposing the oocyte to 5 mM NH₄Cl at pH 6.5 caused a slower and smaller decrease in pH_i, and the depolarization of the cell was significantly inhibited (segment de). The rate of pH_i decrease induced by NH₄Cl was −39 × 10⁻⁴ pH units/s at pH_o 7.5 but only −15 × 10⁻⁴ pH units/s at pH_o 6.5.

Similar experiments were also conducted on H₂O-injected oocytes. As shown in Fig. 7, 5 mM NH₄Cl in the bath at pH 7.5 caused a small change in pH_i and V_m as shown previously (segment ab). Removal of NH₃/NH₄⁺ from the bath caused pH_i and V_m to recover (segment bc). Switching pH_o to 6.5 did not significantly alter pH_i but slightly depolarized the cell (segment cd). Exposing the oocyte again to 5 mM NH₄Cl but at pH 6.5 caused a smaller change of pH_i, at a slower rate, and significantly blocked the depolarization of the cell (segment de). Removal of bath NH₃/NH₄⁺ fully reversed these changes (segment ef). In five paired experiments, 5 mM NH₃/NH₄⁺ at bath pH of 7.5 decreased pH_i by 0.1 ± 0.02 pH units at a rate of −10 ± 1.6 × 10⁻⁴ pH units/s and V_m depolarized by 43 ± 5.6 mV. On the other hand, at bath pH of 6.5, 5 mM NH₃/NH₄⁺ hardly decreased pH_i (0.005 ± 0.0008 pH units at a rate of −2 ± 1.5 × 10⁻⁴ pH units/s) or caused any depolarization of V_m (ΔV_m = 2 ± 1.6 mV). These data confirm the results based on current measurements that NH₄⁺ transport in oocytes is inhibited by acidic pH_o. These data are summarized in Fig. 8.

Fig. 7.

Effect of extracellular acidification on NH₄⁺-induced pH_i and V_m changes in H₂O-injected oocytes. Bath NH₄Cl (5 mM) at pH 7.5 caused a decrease in pH_i at a rate of −14 × 10⁻⁴ pH units/s and depolarized the cell (segment ab). These changes were reversed by removal of NH₄Cl from the bath (segment bc). Switching bath pH to 6.5 did not affect pH_i and only slightly depolarized the oocyte (segment cd). Exposing the oocyte to 5 mM NH₄Cl at pH 6.5 decreased pH_i at a rate of −6 × 10⁻⁴ pH units/s and only slightly depolarized the cell (segment de). These changes were reversed when NH₄Cl was washed out from the bath (segment ef).

Fig. 8.

Summary graph comparing the effects of extracellular acidification on NH₄⁺-induced pH_i and V_m changes in Rhbg-expressing and H₂O-injected oocytes. Lowering bath pH significantly inhibited the NH₃/NH₄⁺-induced changes in oocytes expressing Rhbg (left) and H₂O-injected oocytes (right). *Statistical significance between pH 7.5 and pH 6.5.

The above data indicate that pH_o clearly affects NH₄⁺ transport in oocytes in general and by Rhbg as well. In the next set of experiments, we investigated the role of pH_i in affecting NH₄⁺ transport.

Sensitivity to intracellular pH: effects of intracellular acidification on NH₄⁺-induced current.

The data presented so far demonstrated that, in control oocytes as well as those expressing Rhbg, NH₄⁺ transport is very sensitive to changes in pH_o. In the next set of experiments, we investigated whether NH₄⁺ transport was affected by changes in pH_i. To do so, we acidified the intracellular medium by exposing the oocyte to 20 mM butyrate. In previous studies (20, 22) and as demonstrated below, bath butyrate decreased pH_i by ∼0.2–0.3 pH units. Accordingly, we measured the NH₄⁺-induced current at steady-state pH_i and at acidic pH_i (in the presence of butyrate).

In oocytes expressing Rhbg (Fig. 9A), exposing the oocyte to NH₄Cl (5 mM) caused the usual inward current (segment ab), which readily recovered upon removal of NH₄Cl from the bath (segment bc). At point c, the bath solution was switched to a solution containing 20 mM butyrate at pH 7.5. This is expected to lower pH_i as described above; however, there was no effect on the current, with the voltage clamped at −60 mV (segment cd). In the presence of butyrate, the oocyte was exposed again to 5 mM NH₄Cl, but this time the NH₄⁺-induced current was completely blocked (segment de). These steps were subsequently reversed: NH₄Cl was first removed from the bath with no effect on current (segment ef), and then butyrate was removed and current did not change as well (segment fg). At point g, presumably pH_i went back to steady state; NH₄Cl was applied again to the bath, resulting in an inward current (segment gh) as observed in the first pulse (compare with segment ab). The NH₄⁺-induced current averaged −84 ± 6.1 nA (n = 11) at steady-state pH_i conditions but was only −8 ± 3.1 nA (n = 6; P < 0.05) at low pH_i (in the presence of butyrate). These results indicate that intracellular acidification inhibited NH₄⁺-induced current in oocytes expressing Rhbg.

Fig. 9.

Effect of intracellular acidification on NH₄⁺-induced current. A: in oocytes expressing Rhbg, NH₄Cl (5 mM) caused an inward current (segment ab) that readily reversed upon removal of NH₄Cl from the bath (segment bc). Butyrate (But; 20 mM) in the bath did not cause any change in whole cell current (segment cd). Butyrate decreased pH_i by ∼0.3 units (see Fig. 12). Subsequent additions of NH₄Cl (5 mM) to the bath in the continued presence of butyrate did not induce any significant inward current (segment de). At point e, NH₄Cl was removed from the bath; then at point f butyrate was also removed, with no effects on current. Readdition of NH₄Cl to the bath in the absence of butyrate caused an inward current (segment gh) similar to the 1st pulse (segment ab). B: in H₂0-injected oocytes, similar effects of butyrate on NH₄Cl-induced current were observed. NH₄Cl (5 mM) in the absence of butyrate caused an inward current (segments ab and gh). NH₄Cl (5 mM) in the presence of butyrate did not induce any inward current (segment bc). C: summary of the effects of intracellular acidification (induced by 20 mM butyrate) on NH₄⁺-induced current in Rhbg-expressing and H₂O-injected oocytes. In both cases intracellular acidification significantly inhibited NH₄⁺-induced current. *Statistical significance (P < 0.05).

Similar experiments were also conducted on H₂O-injected oocytes. As shown in Fig. 9B, 5 mM NH₄Cl in the bath caused the usual inward current (segment ab), which recovered upon return of bath solution to standard HEPES (segment bc). At point c, butyrate (20 mM) was added to the bath, causing no change in whole cell current (segment cd). Bath NH₄Cl in the presence of butyrate (and presumably at lower pH_i) caused no change in current (segment de). These steps were then sequentially reversed: NH₄Cl was first removed (segment ef) and then butyrate (segment fg), causing no change in current in both cases. The last step was a repeat of the initial NH₄⁺ control pulse: NH₄Cl added to the bath in the absence of butyrate caused an inward current (segment gh) as observed initially (compare with segment ab). The NH₄⁺-induced change in current averaged −43 ± 8.4 nA (n = 7) in the absence of butyrate but was only −12 ± 7.6 nA (n = 5) at low pH_i (in the presence of butyrate).

This experiment yielded results that are qualitatively similar to those obtained in oocytes expressing Rhbg. These results demonstrate that intracellular acidification inhibits NH₄⁺-induced current in control oocytes also. The data on Rhbg and H₂O-injected oocytes are summarized in Fig. 9C.

pH Dependence of MA/MA⁺ Transport by Rhbg

We have thus far demonstrated that NH₄⁺ transport in oocytes expressing Rhbg as well as in H₂O-injected oocytes is pH dependent. In principle, NH₄⁺ transport seems to be inhibited by low pH (extracellular as well as intracellular) and activated by alkaline pH_o. It is evident that Rhbg transport of NH₄⁺ is clearly affected by pH changes, yet the background transport of NH₄⁺ in H₂O-injected (control) oocytes was also pH sensitive, although with some quantitative differences. Therefore the next series of experiments were conducted to isolate the pH sensitivity of Rhbg from that of native transport in the oocyte preparation. To do so, we measured MA/MA⁺-induced current and pH_i changes while varying bath pH (pH_o) or pH_i.

Sensitivity of MA/MA⁺ current to extracellular pH.

In the first set of experiments, we measured the MA⁺-induced inward current (clamp voltage at −60 mV) at normal bath pH (pH_o) of 7.5, at alkaline pH_o of 8.2, and at acidic pH_o of 6.5. In oocytes expressing Rhbg (Fig. 10A), exposure of the cell to MA/MA⁺ (5 mM) at bath pH of 7.5 caused an inward current of −35 ± 5.9 nA (n = 4; segment ab), which was readily reversed upon removal of MA/MA⁺ (segment bc). At point c, bath pH was raised to 8.2, which did not cause any change in the whole cell current (segment cd). Exposing the oocytes again to MA/MA⁺ (5 mM) at bath pH of 8.2 caused an inward current (segment de) that was substantially larger than that at bath pH of 7.5. In four experiments on oocytes expressing Rhbg, MA⁺-induced current at pH_o 8.2 averaged −147 ± 4.4 nA. The MA⁺-induced current was reversed upon removal of bath MA/MA⁺ (segment ef). Switching bath pH to 6.5 did not cause any change in the clamped current (segment fg). Exposing the oocytes to MA/MA⁺ (5 mM) at pH_o 6.5 did not induce any current (segment gh). This is in contrast to the usual inward current induced by MA⁺ at normal pH or the enhanced MA⁺-induced current at alkaline pH.

Fig. 10.

Effect of pH_o on methyl ammonium (MA⁺)-induced current. A: in oocytes expressing Rhbg, methyl amine (MA)/MA⁺ (5 mM) at pH_o 7.5 caused an inward current (segment ab) that readily reversed upon removal of MA/MA⁺ from the bath (segment bc). Raising bath pH to 8.2 did not cause any change in current (segment cd). MA/MA⁺ at pH_o 8.2 caused a significantly larger inward current (segment de) that recovered upon removal of MA/MA⁺ (segment ef). Lowering bath pH to 6.5 did not cause any change in current (segment fg). MA/MA⁺ at pH_o 6.5 did not cause any inward current (segment gh). No changes in current were detected in subsequent removal of MA/MA⁺ at pH_o 6.5 (segment hi) or upon restoring bath pH to 7.5 (at point i). B: in H₂O-injected oocytes, MA/MA⁺ (5 mM) did not induce any current at pH 7.5 (segment ab), or at pH 8.2 (segment de) or pH 6.5 (segment gh). Changing bath pH from 7.5 to 8.2 or to 6.5 also did not cause any changes in current.

In contrast, in H₂O-injected oocytes (Fig. 10B) exposure to MA/MA⁺ (5 mM) in the bath did not cause any inward current at pH 7.5 (segment ab) as observed above. Similarly, exposure to MA/MA⁺ did not induce any current at pH_o 8.2 (segment de) or at pH_o 6.5 (segment gh). Six similar experiments were conducted.

These data indicate that electrogenic MA⁺ transport by Rhbg is sensitive to changes in external pH. MA⁺-induced currents are enhanced by alkaline external pH and inhibited by acidic bath pH. These effects occur in oocytes expressing Rhbg and are completely absent in H₂O-injected oocytes.

Sensitivity of MA/MA⁺ current to intracellular pH.

To investigate the effect of pH_i on Rhbg transport of MA/MA⁺, we lowered pH_i by exposing the oocyte to a bath solution containing 20 mM butyrate. As described above, in earlier studies we showed that 20 mM butyrate decreased pH_i by ∼0.3 pH units. In this study on oocytes expressing Rhbg (Fig. 11A), we measured the MA⁺-induced current at normal bath pH (7.5), which averaged −52 ± 6.3 nA (n = 3) (segment ab). Removal of MA/MA⁺ from the bath reversed this effect (segment bc). The same oocytes were then superfused by a solution containing 20 mM butyrate, with no effect on current (segment cd). After a period of ∼3 min (during which pH_i usually decreases to a stable, more acidic value), the oocytes were exposed to 5 mM MA/MA⁺ in the continued presence of butyrate. In oocytes expressing Rhbg, exposure to MA/MA⁺ in the presence of butyrate did not induce any inward current (segment de).

Fig. 11.

Effect of intracellular acidification [induced by 20 mM butyrate (Butyr)] on MA⁺-induced current. A: in oocytes expressing Rhbg, MA/MA⁺ (5 mM) caused an inward current (segment ab) that readily reversed upon washout of MA/MA⁺ from the bath (segment bc). pH_i was lowered by exposing the oocyte to butyrate (20 mM), which did not cause any change in whole cell current (segment cd). In the presence of butyrate, MA/MA⁺ in the bath did not cause any inward current (segment de). MA/MA⁺ was then removed from the bath (segment ef), followed by butyrate (at point f) with no effects on current. B: in H₂O-injected oocytes, MA/MA⁺ (5 mM) in the bath did not induce any inward current in the absence of butyrate (segment ab) or in its presence (segment de). C: summary graph indicating the effect of intracellular acidification (lowered by butyrate) on MA⁺-induced current in Rhbg-expressing and H₂O-injected oocytes. Decreasing pH_i completely blocked the MA⁺-induced current in oocytes expressing Rhbg. In H₂O-injected oocytes MA⁺ did not induce any current at steady-state pH_i or when pH_i was lowered by butyrate. *Statistical significance (P < 0.01).

Similar experiments were also conducted on H₂O-injected oocytes. In those experiments (Fig. 11B), MA/MA⁺ did not elicit any significant inward current in the absence (segment ab) or presence (segment de) of butyrate. The effects of pH_i acidification on MA⁺-induced current in Rhbg and H₂O-injected oocytes are summarized in Fig. 11C.

Effect of intracellular acidification on MA-induced pH_i changes.

The possible inhibition of MA/MA⁺ transport by acidic shifts in pH_i was further investigated. To do so, we measured MA/MA⁺-induced changes in V_m and pH_i at steady state as well as acidic pH_i. As shown in Fig. 12, exposing an oocyte expressing Rhbg to MA/MA⁺ (5 mM) caused the usual rapid pH_i increase and significant depolarization of the cell (segment ab). In four experiments, MA/MA⁺ caused pH_i to increase by 0.19 ± 0.03 pH units at a rate of 62 ± 12 × 10⁻⁴ pH units/s and V_m depolarized by 48 ± 2.4 mV. pH_i and V_m fully recovered upon removal of MA/MA⁺ from the bath (segment bc). At point c, exposing the oocytes to 20 mM butyrate in the bath caused a decrease in pH_i by 0.33 ± 0.01 (4), and V_m depolarized by 9 ± 3.2 mV (segment cd). In the continued presence of bath butyrate, exposing the oocytes to MA/MA⁺ (5 mM) caused pH_i to increase by 0.20 ± 0.04 pH units at a rate of 45 ± 8.8 × 10⁻⁴ pH units/s (segment de). However, it is important to note that in the presence of butyrate MA/MA⁺ did not cause any depolarization but rather a small hyperpolarization of −4 ± 1.1 mV (segment de). The MA/MA⁺-induced changes in V_m and pH_i were completely reversed upon removal of MA/MA⁺ from the bath (segment ef). Superfusing the oocytes with control standard HEPES solution (removal of butyrate) resulted in full recovery of pH_i and V_m to their initial steady-state values (segment fg). It is noteworthy that while MA/MA⁺ still induced a pH_i increase at low pH_i, the V_m changes were completely blocked. This suggests that in oocytes expressing Rhbg the electrogenic component of MA/MA⁺ transport was blocked at low pH_i. This is consistent with the current measurements shown above (see Fig. 11). The results of this and similar experiments on oocytes expressing Rhbg are summarized in Fig. 13.

Fig. 12.

Effect of intracellular acidification on MA/MA⁺-induced changes in pH_i and V_m in oocytes expressing Rhbg. Exposing the oocyte to MA/MA⁺ (5 mM) caused an increase in pH_i at a rate of 58 × 10⁻⁴ pH units/s and depolarized the cell (segment ab). These changes were reversed upon washout of MA/MA⁺ from the bath (segment bc). Exposing the oocyte to 20 mM butyrate caused significant intracellular acidification of 0.3 pH units and slightly depolarized the oocyte (segment cd). At low pH_i subsequent addition of MA/MA⁺ to the bath still caused an increase in pH_i at a rate of 51 × 10⁻⁴ pH units/s, but there was a small hyperpolarization of the cell (segment de). These changes were fully reversed upon removal of MA/MA⁺ (segment ef) and then butyrate (segment fg). Bath pH was maintained at 7.5 throughout.

Fig. 13.

Summary graph indicating the effect of low pH_i on MA/MA⁺-induced pH_i and V_m changes in oocytes expressing Rhbg. Lowering pH_i did not inhibit the MA/MA⁺-induced increase in pH_i but completely blocked the change in V_m.

For comparison, similar experiments were conducted on H₂O-injected oocytes. As shown in Fig. 14, exposure of control oocytes to 5 mM MA/MA⁺ did not cause any significant changes in pH_i and V_m (segment ab), after which MA/MA⁺ was removed from the bath (segment bc). At point c, 20 mM butyrate in the bath caused a substantial decrease in pH_i of 0.33 ± 0.03 pH units (3) and a small depolarization of the cell of 8 ± 0.5 mV (segment cd). MA/MA⁺ (5 mM) in the presence of butyrate did not elicit any pH_i increase, and V_m continued to depolarize slowly (segment de). At point e, MA/MA⁺ was removed from the bath, with no significant changes. pH_i and V_m fully recovered when butyrate was removed from the bath (segment fg). These results confirm that MA/MA⁺ effects on pH_i and V_m are Rhbg specific.

Fig. 14.

Effect of intracellular acidification on MA/MA⁺-induced changes in H₂O-injected oocytes. Exposing control oocytes to 5 mM MA/MA⁺ did not induce any significant changes in pH_i or V_m at steady-state pH_i (segment ab) or when pH_i was lowered by 20 mM butyrate (segment de). Tracing shows that butyrate caused substantial decrease in pH_i (segment cd) and small depolarization of the cell. All changes were reversible.

DISCUSSION

This study examined the sensitivity of Rhbg to pH. To do so, we investigated whether NH₃/NH₄⁺ and MA/MA⁺ transport by Rhbg were affected by changes in pH. Voltage-clamp experiments and pH_i monitoring were conducted to assess transport in Xenopus oocytes expressing Rhbg. The sensitivity of MA/MA⁺ and NH₃/NH₄⁺ transport to pH was examined when pH_o was increased or decreased and when pH_i was acidified. Our results indicate a significant sensitivity of Rhbg to changes in pH. In general, transport by Rhbg was stimulated when pH_o was alkaline. However, transport by Rhbg was inhibited when pH_o or pH_i was acidified.

Few studies have addressed the role of pH in regulating transport by members of the Rh family. Using micromolar concentrations of MA/MA⁺ in oocytes expressing Rhbg, Ludewig (12) reported that MA/MA⁺ did not induce any inward current and that uptake of labeled MA/MA⁺ was enhanced with increased pH_o. Even though there was a pH_o stimulation of labeled MA/MA⁺ uptake in H₂O-injected oocytes (yet smaller than in Rhbg), it was concluded that Rhbg transport of MA/MA⁺ was electroneutral and that Rhbg acted as an NH₄⁺/H⁺ (or MA⁺/H⁺) exchanger. Mak et al. (14) also used uptake studies of labeled MA/MA⁺ and demonstrated dependence of uptake on pH_o. That study demonstrated that the pH-enhanced MA/MA⁺ uptake could not be fully explained by an effect on electroneutral MA uptake. In a more thorough examination of the pH effect, the study maintained a constant electroneutral MA concentration while pH_o and total MA/MA⁺ concentrations were varied. This study also reported that uptake of labeled MA/MA⁺ was not affected by manipulating V_m in voltage-clamp experiments. Both studies propose that Rhbg acts as an electroneutral MA⁺/H⁺ (or NH₄⁺/H⁺)exchanger. Our data do not support that Rhbg is behaving as an NH₄⁺/H⁺ exchanger.

Electrogenic NH₄⁺ and MA⁺ Transport by Rhbg

We have previously demonstrated that NH₄⁺ transport by Rhbg is electrogenic because there is a direct and proportional relationship between NH₄⁺-induced inward current and the NH₄⁺ gradient. This relationship is saturable. It is also clear that there is an endogenous electrogenic NH₄⁺ transport in oocytes, as shown by other studies as well (4, 6, 21, 24). However, the magnitude of this transport is enhanced severalfold by expression of Rhbg. Whereas it is difficult to disprove that expression of Rhbg might activate endogenous transport, it is more likely that a de novo transport by Rhbg is expressed even though it may resemble endogenous NH₄⁺ transport. It is interesting to note that Xenopus oocytes do express Rh-related proteins (7, 23).

Our data also indicate that a component of MA/MA⁺ transport by Rhbg is electrogenic. In our experiments, MA/MA⁺ caused an inward current and depolarization of the cell, much like NH₄⁺. This effect was completely absent in H₂O-injected oocytes, suggesting that 1) there is no significant endogenous electrogenic MA⁺ transport in oocytes and 2) MA and/or MA⁺ are good substrates for Rhbg. We used a concentration of 5 mM MA/MA⁺ in most experiments; however, a MA⁺-induced current and depolarization were clearly evident in response to 1 mM MA/MA⁺ (18). In contrast, Ludewig (12) reported no MA⁺-induced current. However, in his experiments, micromolar concentrations of MA⁺ were used. This could be explained by a subthreshold effect of MA⁺ rather than the absence of electrogenic transport.

pH Dependence of NH₄⁺ Transport by Rhbg

In addressing the role of pH_o in affecting transport of NH₄⁺ by Rhbg, our data indicate that alkaline pH_o causes 1) an increase in NH₄⁺-induced current, 2) a faster rate of NH₄⁺-induced changes in pH_i (dpH_i/dt), 3) a bigger NH₄⁺-induced decrease in pH_i (ΔpH_i), and 4) no apparent change in NH₄⁺-induced depolarization. These results indicate that in oocytes expressing Rhbg extracellular alkalinization activates electrogenic NH₄⁺ influx. However, extracellular alkalinization promoted similar changes in NH₄⁺ transport in H₂O-injected oocytes. It should be noted, however, that the voltage and pH changes were not as large. In other words, endogenous NH₄⁺ transport was also enhanced by extracellular alkalinization.

An unexpected observation was that the percent increase in NH₄⁺-induced current at pH 8.2 seemed to be more pronounced in H₂O-injected oocytes compared with oocytes expressing Rhbg. The reasons behind the difference in response are unclear, especially since the nature of the endogenous NH₄⁺ current in H₂O-injected oocytes is unknown. However, in oocytes expressing Rhbg the NH₃/NH₄⁺-induced current and pH_i changes are determined by how the relative flux of NH₃ to NH₄⁺ (J_NH3/J_NH4⁺) changes (17). For example, if J_NH4⁺ and J_NH3 change significantly but proportionally, then the change in current (and ΔpH_i) would be less than if J_NH4⁺ changed, even moderately, but without a matching change in J_NH3. It is therefore possible that at alkaline bath pH, the relative change in J_NH3/J_NH4⁺ is different for Rhbg than for H₂O-injected oocytes. Our results on MA/MA⁺, where there is no significant endogenous transport, are consistent with this possibility.

The effect of extracellular acidification on NH₄⁺ transport in oocytes expressing Rhbg was in the opposite direction. In oocytes expressing Rhbg, acidic pH_o caused 1) complete inhibition of NH₄⁺-induced current, 2) inhibition of NH₄⁺-induced rate of pH_i decrease, 3) smaller NH₄⁺-induced pH_i decrease (ΔpH_i), and 4) inhibition of NH₄⁺-induced change in V_m. These results indicate that extracellular acidification inhibits electrogenic NH₄⁺ transport by Rhbg. However, acidic pH_o caused similar but smaller effects on NH₄⁺ transport in H₂O-injected oocytes.

Because the effect of pH_o on NH₄⁺ transport was qualitatively similar in H₂O-injected and Rhbg oocytes, two possibilities could be proposed. First, expression of Rhbg upregulates endogenous NH₄⁺ transport with no distinct NH₄⁺ pathway through Rhbg. Alternatively, NH₄⁺ transport by Rhbg is distinct from that of endogenous NH₄⁺ in control oocytes even though they share several similar characteristics, yet both are activated by extracellular alkalinization.

We propose the latter hypothesis as the working model for NH₄⁺ transport by Rhbg. This model is supported by the following observations: 1) If endogenous NH₄⁺ transport was inhibited by a decrease in pH_o (or activated by an increase in pH_o), this does not exclude the possibility that transport by Rhbg may be affected similarly. Given the fact that Xenopus oocytes possess membrane proteins belonging to the Rh family, endogenous NH₄⁺ transport may resemble that of Rhbg. 2) Amiloride was able to inhibit electrogenic NH₄⁺ transport in oocytes expressing Rhbg but not in H₂O-injected oocytes (18). This observation does not favor an upregulation of endogenous NH₄⁺ transport by Rhbg expression. 3) MA/MA⁺ acts as specific substrate for Rhbg, causing intracellular changes that are different from those in H₂O-injected oocytes.

The dependence of NH₄⁺ transport on changes in pH_o raises two possibilities. The first is that NH₄⁺ transport could be driven by the H⁺ gradient. This possibility is favored by the studies proposing that Rhbg functions as NH₄⁺/H⁺ exchanger (12–14, 27). Alternatively, Rhbg may be directly affected by pH, possibly through a charge or allosteric effect resulting in inhibition by low pH and activation by an increase in pH.

To distinguish between these two possibilities, we investigated the dependence of NH₄⁺ transport by Rhbg on pH_i. If Rhbg is an NH₄⁺/H⁺ exchanger driven by the H⁺ gradient, then raising intracellular [H⁺] should also lead to activation of transport. In our experiments, lowering pH_i by exposing oocytes to 20 mM butyrate caused 1) inhibition of NH₄⁺ current, 2) inhibition of NH₄⁺-induced ΔpH_i, 3) inhibition of NH₄⁺-induced rate of pH_i decrease, and 4) inhibition of NH₄⁺-induced depolarization. These observations do not favor NH₄⁺/H⁺ exchange but rather indicate that Rhbg is pH sensitive. It is inhibited by low pH, intracellular or extracellular, and activated by extracellular alkalinization.

The implications of inhibiting transport by Rhbg at low pH are difficult to interpret. NH₃/NH₄⁺ transport in the collecting duct increases during acidosis. Therefore, if Rhbg (and Rhcg for that matter) is a major route of NH₃/NH₄⁺ excretion, it is expected that transport would be enhanced rather than inhibited at low pH. To resolve this apparent disparity, at least two issues need to be addressed. First, a profile of Rhbg dependence over a range of pH values would determine how activity of Rhbg changes at different pH values. Second, the effects of low pH on NH₄⁺ versus NH₃ transport need to be determined. Our data clearly indicate that electrogenic NH₄⁺ transport was inhibited at low pH. It is not clear, however, whether NH₃ transport (and consequently net NH₃/NH₄⁺ transport) was similarly affected. In fact, the experiments with MA/MA⁺ showed that electroneutral MA transport was not significantly altered. A role of Rhbg in acidosis is demonstrated in studies where tissue-specific deletion of Rhbg in mouse CD caused more severe acidosis and impaired NH₄⁺ excretion in response to acid loading (2).

pH Dependence of MA/MA⁺ Transport by Rhbg

Transport of MA/MA⁺ by Rhbg provides additional insight into the properties of this mechanism. Our experiments indicate that expressing Rhbg in oocytes promotes transport of electrogenic MA⁺ and electroneutral MA. The former (MA⁺) appears to be transported by Rhbg much like NH₄⁺. However, there was no evidence of electrogenic MA⁺ transport in H₂O-injected oocytes where MA⁺ did not induce any current or depolarization of the cell. Thus in electrical measurements MA⁺ is very useful as a good substrate for Rhbg. The fact that charged MA⁺ induces electrical changes similar to NH₄⁺ in Rhbg oocytes but not in H₂O-injected oocytes argues against the possibility that enhanced NH₄⁺ conductance and current by Rhbg is caused by activating endogenous NH₄⁺ transport.

On the other hand, MA/MA⁺ transport by Rhbg induced an increase in pH_i consistent with electroneutral transport of MA by Rhbg. This is unique in the oocyte, especially that NH₃ hardly causes any detectible pH_i alkalinization. Moreover, MA-induced alkalinization in H₂O-injected oocytes is minimal compared with that in oocytes expressing Rhbg. One likely possibility is that Rhbg may promote MA transport in addition to the charged MA⁺. The present study cannot distinguish between direct transport of MA and an enhanced membrane permeability of the neutral molecule by the oocytes upon expression of Rhbg. One observation in favor of direct transport of MA/MA⁺ by Rhbg is that amiloride inhibited MA-induced alkalinization as well as MA⁺-induced current and depolarization. However, inhibition of MA-induced alkalinization by low pH_i was less pronounced.

A recent study (10) resolved the molecular structure of AmtB, a bacterial homolog of Rh glycoproteins from Escherichia coli, by X-ray crystallography at 1.35-Å resolution. On the basis of molecular analysis of this structure, a model was proposed in which a 20-Å-long hydrophobic pore serves as a channel that has four NH₃ or NH₄⁺ binding sites. The first site facing the extracellular medium serves as a vestibule that recruits NH₃/NH₄⁺ binding predominantly as an charged cationic NH₄⁺. Once NH₄⁺ is recruited, H⁺ is released extracellularly and NH₃ is conducted through the hydrophobic pore favored by molecular interactions that lower pK_a in the pore. On the intracellular side, NH₃ recruits an intracellular H⁺ and is released as NH₄⁺. Of the four NH₃/NH₄⁺ binding sites, only the first at the entry site accepts a fully protonated NH₄⁺. The other three sites are occupied by fully deprotonated NH₃ (8). According to this model, transport is essentially pH sensitive. As such, decreasing pH_o is thought to lower the rate of transport by opposing the proton release from NH₄⁺. The opposite effect is expected by raising pH_o. These observations are consistent with our results about the effect of bath pH on NH₄⁺ and MA⁺ transport. On the other hand, decreasing pH_i should not lead to inhibition of transport. A decrease in pH_i is expected to favor reprotonation of NH₃ and therefore acceleration of transport. In our experiments this was not the case, but rather a decrease in pH_i caused an inhibition of NH₄⁺ transport by Rhbg. Whether these differences are unique to Rhbg compared with AmtB will be addressed in future studies.

The model suggesting Rhbg to be an electroneutral NH₄⁺/H⁺ exchanger was largely based on uptake studies of labeled MA/MA⁺ and its sensitivity to changes of pH_o. It is important to note that in those studies it was not possible to distinguish between electroneutral MA and electrogenic MA⁺ transport, both of which seem to be promoted by Rhbg expression. Moreover, in those studies only pH_o could be manipulated, and therefore inhibition by low pH_i (which does not favor the exchange model) could not be examined.

In summary, our study demonstrated that transport of NH₄⁺ and MA/MA⁺ by Rhbg is pH sensitive. Electrogenic NH₄⁺ and MA⁺ transport are stimulated by alkaline pH_o but inhibited by acidic pH_i or pH_o. Electroneutral MA transport by Rhbg is likely but seems to be less sensitive to pH changes. An H⁺ gradient driving transport by Rhbg (as suggested by an exchange model) cannot be fully explained by our data. On the other hand, direct effects of pH on Rhbg are very likely.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-62295 and American Heart Association Southern Affiliate Grant 0255258B.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).