NH3 is involved in the NH4+ transport induced by the functional expression of the human Rh C glycoprotein.

Renal ammonium (NH3 + NH4+) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow NH4+ transmembrane translocation without high specificity NH4+, but it is still debated whether NH3, and more generally, gas, may be transported by transmembrane proteins. The human Rh glycoproteins have been proposed to mediate ammonium transport. Transport of NH4+ and/or NH3 by the epithelial Rh C glycoprotein (RhCG) may be of physiological importance in renal ammonium excretion because RhCG is mainly expressed in the distal nephron. However, RhCG function is not yet established. In the present study, we search for ammonium transport by RhCG. RhCG function was investigated by electrophysiological approaches in RhCG-expressing Xenopus laevis oocytes. In the submillimolar concentration range, NH4Cl exposure induced inward currents (IAM) in voltage-clamped RhCG-expressing cells, but not in control cells. At physiological extracellular pH (pHo) = 7.5, the amplitude of IAM increased with NH4Cl concentration and membrane hyperpolarization. The amplitude of IAM was independent of external Na+ or K+ concentrations but was enhanced by alkaline pHo and decreased by acid pHo. The apparent affinity of RhCG for NH4+ was affected by NH3 concentration and by changing pHo, whereas the apparent affinity for NH3 was unchanged by pHo, consistent with direct NH3 involvement in RhCG function. The enhancement of methylammonium-induced current by NH3 further supported this conclusion. Exposure to 500 microm NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and NH4+ enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH3 and NH4+ transport.

) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow NH 4 ؉ transmembrane translocation without high specificity for NH 4 ؉ , but it is still debated whether NH 3 , and more generally, gas, may be transported by transmembrane proteins. The human Rh glycoproteins have been proposed to mediate ammonium transport. Transport of NH 4 ؉ and/or NH 3 by the epithelial Rh C glycoprotein (RhCG) may be of physiological importance in renal ammonium excretion because RhCG is mainly expressed in the distal nephron. However, RhCG function is not yet established. In the present study, we search for ammonium transport by RhCG. RhCG function was investigated by electrophysiological approaches in RhCG-expressing Xenopus laevis oocytes. In the submillimolar concentration range, NH 4 Cl exposure induced inward currents (I AM ) in voltage-clamped RhCG-expressing cells, but not in control cells. At physiological extracellular pH (pH o ) ‫؍‬ 7.5, the amplitude of I AM increased with NH 4 Cl concentration and membrane hyperpolarization. The amplitude of I AM was independent of external Na ؉ or K ؉ concentrations but was enhanced by alkaline pH o and decreased by acid pH o . The apparent affinity of RhCG for NH 4 ؉ was affected by NH 3 concentration and by changing pH o , whereas the apparent affinity for NH 3 was unchanged by pH o , consistent with direct NH 3 involvement in RhCG function. The enhancement of methylammonium-induced current by NH 3 further supported this conclusion. Exposure to 500 M NH 4 Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH 3 and NH 4 ؉ enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH 3 and NH 4 ؉ transport.
The human Rh family is composed of five known proteins: RhD, RhCE, RhAG, RhBG, and RhCG. Whereas RhD, RhCE, and RhAG proteins are expressed in erythroid cells, RhBG and RhCG proteins are expressed in epithelial tissues. Northern blot analyses have shown that RhBG is expressed mainly in the kidney and liver (1) and that RhCG is expressed mainly in the testis and kidney (2,3). Rh proteins share homologies with the Mep/Amt family from yeasts, bacteria, and plants (4,5). Despite numerous studies implicating Mep/Amt proteins in ammonium transport (6,7), the transported substrate is still debated. Recent studies report, on one hand, that AmtB protein facilitates NH 3 diffusion across the cytoplasmic membrane of Salmonella typhimurium (8) and, on the other hand, that LeAMT1;1 acts as an NH 4 ϩ uniport after its functional expression in Xenopus laevis oocytes (9).
Consistent with the involvement of Rh proteins in transmembrane ammonium transport, yeasts deficient in endogenous ammonium transport system (⌬Mep Saccharomyces cerevisiae) are enabled to grow in a low ammonium-containing medium when transformed by RhAG or by RhCG (3). Based on their finding that Mep and Amt are NH 3 channels, Soupene et al. (10,11) raised the hypothesis that Rh proteins are involved in gas transport rather than in ionic transport. However, Westhoff et al. (12) concluded that RhAG mediates an electroneutral ionic exchange of NH 4 ϩ for H ϩ after its functional expression in X. laevis oocytes. To our knowledge, the mechanistic properties of the human proteins RhBG and RhCG are not yet established. These proteins may have an important role in acid-base balance because ammonium excretion by the kidney plays a major role in acid excretion.
The aim of our study was to functionally express RhCG in X. laevis oocytes and investigate whether RhCG is involved in ammonium 1 (NH 4 ϩ and/or NH 3 ) transport. Exposure of voltageclamped RhCG-expressing cells to submillimolar concentrations of NH 4 Cl ([NH 4 Cl]) induced inward currents (I AM ). The amplitude of I AM increased with [NH 4 Cl] and membrane hyperpolarization, consistent with an NH 4 ϩ -related current. However, the amplitude of I AM was strongly sensitive to changes in extracellular pH (pH o ) of Ϯ0.5 pH unit (pH U), 2 an experimental maneuver that only slightly changes [NH 4 ϩ ] but substantially affects [NH 3 ]. At alkaline but not physiological pH o , the enhancement of methylammonium-induced current by micromolar [NH 4 Cl] further supports the requirement of the neutral form for NH 4 ϩ /methylammonium transport in RhCG-expressing oocytes. Exposure to 500 M NH 4 Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH 3 and NH 4 ϩ influx into the cell. These results are consistent with RhCG-induced NH 3 1 In the text, "ammonium" is used when ammonia and ammonium ions are not discriminated. The chemical symbols for ammonia (NH 3 ) and ammonium ions (NH 4 ϩ ) are used to distinguish the two forms of ammonium. 2 The abbreviations used are: pH U, pH unit; GFP, green fluorescent protein; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1propanesulfonic acid; MeACl, methylamine chloride.
terminal fusion of the protein RhCG with the green fluorescent protein (GFP; Ref. 13). GFP-RhCG expression was demonstrated by membrane green fluorescence, observable under microscopic control (excitation wavelength, 480 Ϯ 40 nm; emission filter, 505 nm). RhCG cDNA was amplified by PCR from the pRS426-RhCG construct (3) using 5Ј-CTG-CAGCATGGCCTGGAACACCAACCT-3Ј and 5Ј-CTCCTCACCTGCCC-TGGGAGCCTAGGG-3Ј as sense and antisense primers, respectively. The 1466-bp RhCG cDNA was inserted downstream of the GFP coding region into the pQBI25-fC1 vector. The in-frame insertion of RhCG cDNA was confirmed by sequencing. To synthesize cRNA coding for GFP-RhCG fusion protein, the GFP-RhCG cDNA was subcloned into pT7TS plasmid. The pT7TS-GFP-RhCG construct was linearized with SmaI restriction enzyme and transcribed in vitro from the T7 promoter using an mCAP mRNA capping kit. Defolliculated X. laevis oocytes (stage V-VI) were injected with cRNA dissolved in 50 nl of water or with 50 nl of water and then incubated at 18°C (14). All experiments were performed in RhCG-expressing oocytes (oocytes injected with cRNA of GFP-RhCG) versus control oocytes (oocytes injected with water or cRNA of GFP). No difference was observed between control oocytes expressing GFP alone and control oocytes injected with water.
Voltage-Clamp Experiments-Two-electrode voltage-clamp experiments were performed as described previously (15,16). Except where stated, the oocyte membrane potential was held at Vc ϭ Ϫ50 mV. Oocytes were superfused by a control Ringer solution adapted for amphibia (containing 96 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM Hepes, adjusted to pH 7.5 with NaOH) or by up to seven substitution solutions differing from each other by a single parameter. In all experiments, the first substitution was a 500 M NH 4 Cl-containing solution at pH o 7.5. Solutions at pH o ϭ 7.0 or 8.0 were also buffered with Hepes/NaOH. Solutions at pH o ϭ 8.5 were buffered with TAPS/ NaOH (switching from Hepes to TAPS buffer, at identical pH o , had no effect). In Na ϩ -free experiments, NaCl was replaced by an equimolar concentration of choline chloride, and pH was adjusted using Trizma (Tris base). To calculate [NH 3 ] and [NH 4 ϩ ] in ammonium-containing solutions, the pK a was taken as 9.25.
As reported by other groups (9,17), we noticed that in control oocytes, even at low millimolar concentrations, NH 4 Cl (and methylammonium) may induce an inward endogenous current that increases at alkaline pH o (17). This is likely related to the multiple endogenous cationic conductances that are activated by NH 4 Cl in native X. laevis oocyte (18). In the present study, such endogenous currents were detected in H 2 O-injected oocytes upon exposure to [NH 4 Cl] Ն 3 mM, [NH 4 Cl] Ն 1.5 mM, and [NH 4 Cl] Ն 500 M at pH o ϭ 7.0, 7.5, and 8.0, respectively. At these low [NH 4 Cl], the endogenous current amplitude (I endog ) varied from one batch of oocytes to another. Thus, when applying corrections, the NH 4 Cl-induced response in control cells was subtracted from the NH 4 Cl-induced response measured in RhCG-expressing oocytes from the same batch. However, because I endog in oocytes may also vary within the same batch of oocytes, we avoided as far as possible the use of NH 4 Cl concentrations high enough to induce an endogenous response, except when necessary for further characterization of RhCG functional properties.
Intracellular pH Measurements-Intracellular pH (pH i ) and membrane potential (V m ) were simultaneously measured using double-barreled pH-sensitive microelectrodes (filled with the Fluka H ϩ ionophore 95291), as detailed previously (19). The slope, S, of the pH-selective microelectrode was determined before and after each intracellular puncture by measuring the variation of potential induced by a 0.5 pH U change in pH o . In our experiments, S was 55-59 mV/pH U. pH i was calculated using the relationship pH i ϭ pH o Ϫ (V H Ϫ V m )/S, where V H is the proton electrochemical potential difference across the cell membrane.
Except where stated, results were expressed as means Ϯ S.E. (with n ϭ number of oocytes), and the significance of the results was assessed by paired or unpaired Student t test (p Ͻ 0.05 was considered significant).

RESULTS AND DISCUSSION
Ammonium-induced Current in RhCG-expressing Oocytes-In preliminary experimental series, oocytes were injected with 5, 10, 15, 25, or 45 ng of cRNA of GFP-RhCG or GFP or with water. The effect of increasing [NH 4 Cl] (100, 250, 500, and 1000 M; pH o ϭ 7.5) was investigated in voltage-clamped cells. In control oocytes (n ϭ 21), this experimental maneuver was without effect, except occasionally for [NH 4 Cl] ϭ 1000 M (which may induce a small endogenous inward current, up to Ϫ5 nA). In RhCG-expressing oocytes, this experimental ma-neuver induced increasing inward currents, as shown in Fig. 1. In this series, we also measured pH i . Resting pH i was not modified by RhCG expression (pH i ϭ 7.44 Ϯ 0.02 (n ϭ 23) in RhCG-expressing oocytes versus 7.39 Ϯ 0.03 (n ϭ 18) in control oocytes; p ϭ 0.2). This rules out a pH i change as the explanation for I AM measured in RhCG-expressing oocytes. These results are consistent with the hypothesis that RhCG function is related to ammonium transport (3). For further analysis of I AM , oocytes were injected with 10 ng of cRNA of GFP-RhCG (this concentration seems to give the best functional response; see the inset in Fig. 1) or with water.
Specificity of NH 4 Cl-induced Currents-The voltage dependence of I AM in RhCG-expressing oocytes was investigated by applying a [NH 4 Cl] ϭ 500 M pulse at various holding potential values. The resulting current-voltage relationship (Fig. 2) shows that I AM increased with hyperpolarization (inside negative), consistent with the enhancement of net entry of positive charges (namely, NH 4 ϩ ) into RhCG-expressing oocytes. Extrapolating the current-voltage relationship to more depolarized values (which were not experimentally assessed due to the activation of a Na ϩ conductance induced by depolarization; Ref. 20) gives a reversal potential (E rev ) near 0 mV. This E rev value may correspond to the combined equilibrium potential of the main cationic species (Na ϩ ϩ K ϩ ) of NH 4 ϩ or of H ϩ ions (18). Because a major involvement of H ϩ ions was not supported by the pH i stability of RhCC-expressing oocytes upon pH o change (from pH o 7.5 to 8.5, ⌬pH i ϭ 0.02 Ϯ 0.01; p ϭ 0.3; n ϭ 4), we next determined the specificity of the currents to NH 4 ϩ compared with other cationic species.
First, voltage-clamped oocytes (Vc ϭ Ϫ50 mV) were exposed pairwise to a Ringer solution supplemented with 500 M NH 4 Cl or 500 M choline chloride or NaCl. Neither NaCl nor choline chloride induced a current in RhCG-expressing oocytes or control cells (data not shown), whereas I AM was Ϫ13.2 Ϯ 1.1 nA in RhCG-expressing oocytes (n ϭ 8) but was not detectable in H 2 O-injected cells (I AM ϭ 0.1 Ϯ 0.2; n ϭ 4). The non-involvement of Na ϩ ions in RhCG function was further confirmed by measuring in a paired fashion that the current induced by 500 M NH 4 Cl added to a plain Ringer solution or to a Na ϩ -free solution was the same (n ϭ 10; p ϭ 0.3). This also confirms that RhCG does not mediate a nonselective cationic pathway, in which case I AM would be expected to increase under Na ϩ -free conditions.
Second, the discrimination between K ϩ and NH 4 ϩ in RhCGexpressing oocytes was investigated. To this end, the effect of adding 500 M KCl to the Ringer solution was checked. This maneuver did not induce an inward current in RhCG-expressing oocytes, consistent with high selectivity for NH 4 ϩ over K ϩ . This finding is in agreement with studies in S. cerevisiae showing that RhCG did not complement the growth defect of yeast deficient in K ϩ transport (3). In fact, in this series, we observed a slight KCl-induced outward current in all oocytes (ϩ2.1 Ϯ 0.6 nA in RhCG-expressing oocytes and ϩ0.8 Ϯ 0.7 nA in H 2 Oinjected oocytes; n ϭ 8). This is likely related to the properties of the endogenous oocyte Na,K-pump activity, which is highly sensitive to changes in extracellular [K ϩ ] (18), which was increased by 25% in our experiments. The non-involvement of K ϩ in RhCG function was confirmed by measuring in a paired fashion that the current induced by 500 M NH 4 Cl added in a plain Ringer solution or in a K ϩ -free medium was the same (n ϭ 3; p ϭ 0.5).
The above-mentioned results are consistent with the induction (or enhancement) of an ammonium-related, rheogenic process consecutive to RhCG functional expression. This suggests that RhCG has a different function than those proposed for RhAG (the erythroid homologue of RhCG) or for Rh1 protein from Chlamydomonas reinhardtii (11,12). RhAG, after functional expression in X. laevis oocytes, was reported to mediate an electrically silent NH 4 ϩ /H ϩ exchange (12). In that study, the authors proposed that this electroneutral ionic transport mediated by RhAG would not change pH i , despite the RhAGinduced NH 4 ϩ influx into the cell (12). Another group speculated that human Rh proteins mediate CO 2 gas diffusion (11). According to the authors, this hypothesis is supported by their observation that incubation of the green alga C. reinhardtii in a high-CO 2 environment increased the expression of the related RH gene, RH1 (11). Because our study focuses on ammonium transport, we will not discuss our results in the light of putative CO 2 diffusion by RhCG. With regard to NH 3 , which is also a gas, RhCG-mediated transport of this uncharged species cannot account for the observed I AM , at least not in a simple manner. However, the rheogenicity of the ammonium-induced response does not exclude the possibility that NH 3 is transported together with an ion or that NH 3 stimulates an electrogenic ionic transport in RhCG-expressing oocytes. In the experiments reported above, the discrimination observed between NH 4 ϩ and K ϩ in RhCG-expressing oocytes, as well as the ob-servation that Na ϩ , K ϩ , and choline ϩ do not mimic the effect of NH 4 ϩ on membrane current, agrees with results reported in X. laevis oocytes expressing LeAMT1;1, an Amt protein from tomato root hair (9). Our results strongly suggest that, under our experimental conditions, RhCG function is specifically related to ammonium transport.
Effect of Extracellular pH on NH 4 Cl-induced Currents-When it is expressed in X. laevis oocytes, the Amt protein LeAMT1;1 mediates an NH 4 ϩ uniport (9). The NH 4 ϩ currents induced by LeAMT1;1 expression were pH o -independent (9). To determine whether LeAMT1;1 and RhCG have similar functional properties, we looked for the effect of changing pH o on I AM in voltage-clamped oocytes.
We ϩ ] (see Fig. 3). These solutions were buffered to pH 7.0 (Fig. 3A) or pH 8.0 (Fig. 3B). In both series, I AM was the same during exposure of RhCGexpressing oocytes to solutions with different pH o but containing identical [NH 3 ].
The above-mentioned results show a strong effect of changing pH/NH 3 on NH 4 ϩ -induced current in RhCG-expressing oocytes. This is at variance with results reported for LeAMT1;1expressing oocytes (9). In that study, the pH o independence of I AM was invoked as a strong argument to conclude that LeAMT1;1 mediates an NH 4 ϩ uniport, independent of NH 3 (9). Our results also argue against a putative NH 4 ϩ -H ϩ co-transport mediating both NH 4 ϩ and H ϩ influx into the cell. Such a transport system would induce an I AM inward current, but alkaline external pH should reduce I AM (conversely, acidic pH should increase I AM ), whereas we observed the opposite. These results suggest that NH 3 or H ϩ ions are directly involved in RhCG function.
Substrate Dependence of I AM -Discrimination between H ϩ and NH 3 effects is not obvious because of the equilibrium reaction between NH 4 ϩ , NH 3 , and H ϩ . Nonetheless, we attempted to arrive at a better understanding of RhCG function by establishing the concentration dependence of the ammonium-induced currents on each of these three species. To this end, I AM was measured while keeping pH o , [NH 3 ], or [NH 4 ϩ ] constant while varying the other two.
First, RhCG-expressing oocytes were exposed to increasing [NH 4 Cl] at a constant pH o of 7.5 (Fig. 4A) or 7.0 (Fig. 4B). This protocol gives the dependence of I AM on [NH 4 Cl]. This represents, to a first approximation, the I AM dependence on [ NH 4 ϩ ] because NH 4 ϩ concentration is so much higher than that of NH 3 in this pH range. The current-concentration relationships saturated at relatively low substrate concentrations, suggesting carrier (rather than channel) behavior. The apparent affinity for ammonium (thus, in a first approximation, for [NH 4 ϩ ]) appears to be pH o -dependent. This observation is at variance with results reported for the function of RhAG because changes in pH o did not modify the kinetics of ammonium inhibition of methylammonium uptake measured in RhAG-expressing oocytes (12). This finding was taken as an argument against the transport by RhAG of NH 3 or of the neutral form of methylammonium (12). Interestingly, in RhCG-expressing oocytes, the apparent NH 3 affinity (Ϸ8 M) seems to be pH o -independent.
In another experimental series, RhCG-expressing oocytes were exposed to increasing [NH 3 ] at constant [NH 4 ϩ ] (pH o was changing). This protocol was carried out at constant [NH 4 ϩ ] Ϸ 491 M (Fig. 5A) or 246 M (Fig. 5B). Results confirmed that half-maximal I AM was reached for [NH 3 ] Ϸ 8 M (corresponding to different pH o ) as in the previous series (Fig. 4). This finding suggests NH 3 transport by RhCG and/or its direct involvement in I AM .
In a complementary protocol, RhCG-expressing oocytes were exposed to increasing [NH 4 ϩ ] at constant [NH 3 ] (in the same range of pH o values as in the previous protocol, i.e. from pH o 7.0 to 8.0 by increments of 0.2 pH U). This experimental series was performed with [NH 3 ] constant at 8.73 or 4.36 M. Analysis (by analysis of variance) of the results (data not shown) obtained in n ϭ 5-7 oocytes showed that I AM was constant for a given [NH 3 ], despite the 10-fold change imposed in [NH 4 ϩ ] between pH 7.0 and 8.0. These results agree with results from Fig. 3 and suggest either that [NH 3 ] acts as an "on-off switch" for I AM or that NH 4 ϩ was at a saturating concentration. To discriminate between these possibilities, RhCG-expressing oocytes were again exposed to increasing [  cytes, this maneuver induced an endogenous current (I endog ) that increased with increasing pH o , in agreement with a previous report (17). In RhCG-expressing oocytes, this maneuver induced a total current, I TOT , due to I endog ϩ I AM . Because I endog variability appears to be high (especially at alkaline pH o ) not only from one batch of oocytes to another but also within the oocytes of a given batch, we did not calculate I AM from I TOT Ϫ I endog . Fig. 6 shows that [NH 4 Cl] ϭ 5 mM induced significantly higher ammonium-induced current in RhCG-expressing oocytes than in H 2 O-injected oocytes. Results from Fig. 6 are also consistent with an increase of I AM when raising pH o and vice versa. This suggests that extracellular pH influences the maximal NH 4 ϩ -induced current mediated by RhCG. With regard to the results shown in Fig. 4 and Fig. 5, C and D, it may be that the pH o effect modified the value of K m for NH 4 ϩ , decreasing it at alkaline pH o and increasing it at acidic pH o .
The results also agree with the dependence of I AM on the concentrations of both the charged (namely, NH 4 ϩ ) and uncharged (namely, NH 3 ) forms of ammonium in RhCG-expressing oocytes. To better understand the role of NH 3 in NH 4 ϩ transport and RhCG function, we next investigated its effect on the transport of NH 4 ϩ analogues by RhCG and the effect of NH 4 Cl exposure on intracellular pH.
Effect of Neutral Form on Current Induced by Analogues of NH 4 ϩ -To clarify whether, in RhCG function, the neutral form activates an ionic current related to the charged form, we investigated the effect of methylamine (H 3 C-NH 2 /H 3 C-NH 3 ϩ ) on oocytes by checking whether methylamine chloride (MeACl) induces a current in voltage-clamped oocytes. Methylammonium is commonly used as an ammonium analogue. Several studies focusing on Rh, Mep, or Amt proteins showed that it may be transported in place of ammonium (7,9,10,12).
First, we looked for methylammonium-induced current, I MeA , at pH o ϭ 7.5. Because H 3 C-NH 2 /H 3 C-NH 3 ϩ has a higher pK a than NH 3  . At pH o 7.5, the same maneuver was without effect (Fig.  7). This result was expected because at pH o 7.5, in a solution of 50 M NH 4 Cl, the [NH 3 ] is Ͻ1 M, which is very low compared with the measured half-maximal concentration of Ϸ8 M.
Taken together, our results agree with NH 3 involvement in NH 4 ϩ (H 3 C-NH 3 ϩ )-induced current. This finding is specific to RhCG compared with its erythroid homologue. In RhAG-expressing oocytes, methylammonium uptake was reported not to be rheogenic and to be independent of [NH 3 ] (12). In RhCGexpressing oocytes, our results suggest an electrogenic methylammonium influx activated by [NH 3 ].
An ammonium-induced activation of the AE2 exchanger after its functional expression in X. laevis oocyte has been documented and was partly explained by an NH 4 Cl-induced intracellular Ca 2ϩ increase (21). However, a similar explanation cannot account for our results because ammonium-induced activation of AE2 was observed in the presence of [NH 4 Cl] ϭ 10 -20 mM (21), an experimental condition very different from our conditions (stimulation of I MeA was observed using micromolar concentrations of NH 3 /NH 4 ϩ ). Moreover, in our experiments, I MeA stimulation appears to be specifically related to RhCG expression because it was only observed in RhCG-expressing oocytes, not in water-injected oocytes. Interestingly, Rh proteins were proposed be "sensors" and to regulate ion transport, by analogy to MEP2, which was shown to regulate the cell differentiation in S. cerevisiae (3,(22)(23)(24). According to this proposal, RhCG might be a regulator of ammonium transport, an interesting hypothesis for a protein whose function in native tissue is not yet known. Interactions between the epithelial Rh glycoproteins RhCG and RhBG and acid-equivalent transport systems have also been speculated (21,23) due to their localization in native tissue. In mouse distal nephron, AE1 and RhBG are co-localized in the basolateral membrane of type A cells (23), and in the rat and mouse kidneys, RhCG is localized in the apical membrane of tubular cells that are involved in ammonium secretion and in net transepithelial acid-base transport (23,25). However, from our results, we can only conclude that transport of NH 4 ϩ /H 3 C-NH 3 ϩ is stimulated by the neutral form of these compounds in RhCGexpressing oocytes.
In light of these results suggesting that [NH 3 ] stimulates I MeA , we further checked our previous conclusion that RhCG discriminates between NH 4 ϩ and K ϩ (known to be competitive substrates on various transport systems) because the observed lack of K ϩinduced inward current in RhCG-expressing oocytes was obtained using NH 3 -free solutions. Thus, we looked for an induced current when adding 50 M NH 4 Cl to a Ringer solution supplemented with 500 M KCl at pH o ϭ 8.5. This maneuver did not induce an inward current in RhCG-expressing oocytes (p ϭ 0.5; n ϭ 3). This further confirms that RhCG function is specifically related to ammonium (methylammonium) transport.
Effect of NH 4  ured the effect on pH i of exposing oocytes to 500 M NH 4 Cl (pH o ϭ 7.5). Control and RhCG-expressing oocytes exhibited the same resting pH i (pH i ϭ 7.38 Ϯ 0.02 (n ϭ 9) versus 7.39 Ϯ 0.01 (n ϭ 9)), but exposure to NH 4 Cl induced a pH i change (biphasic change) only in RhCG-expressing oocytes, as shown in Fig. 8. In RhCG-expressing oocytes, the initial effect of NH 4 Cl exposure was a slight transient alkalinization (pH i increased to 7.42 Ϯ 0.01, which was significantly different from resting pH i , p Ͻ 0.05), which was not detected in H 2 O-injected oocytes (pH i was stable (7.38 Ϯ 0.02) and was not different from resting pH i , p ϭ 0.3). During NH 4 Cl exposure, pH i significantly decreased to 7.34 Ϯ 0.01 in RhCG-expressing oocytes but was unchanged in control oocytes (7.38 Ϯ 0.03). Using a 20 mM NH 4 Cl-containing solution, we had previously reported that the oocyte membrane is only slightly permeable to NH 3 compared with NH 4 ϩ but that initial NH 3 influx, ⌽ NH 3 , into the cell is reflected by a small but detectable cell alkalinization preceding the cell acidification due to NH 4 ϩ influx, ⌽ NH 4 (18,26). In the present study, application of 500 M NH 4 Cl had no effect on pH i in control oocytes. This indicates that this NH 4 Cl concentration is too low to induce any detectable ⌽ NH 3 or ⌽ NH 4 through the oocyte endogenous membrane pathways for ammonium. Thus, in the presence of 500 M NH 4 Cl, the biphasic pH i change observed in RhCG-expressing oocytes reflects enhanced ⌽ NH 3 and ⌽ NH 4 as compared with control oocytes. One may argue that RhCG expression has simply enhanced endogenous NH 3 and NH 4 ϩ transport in the oocyte. However, the saturation of I AM as a function of [NH 4 Cl] (Fig. 4)   pathways (18), thus supporting our conclusion that RhCG expression induces a heterologous transport of both NH 3 and NH 4 ϩ . Nonetheless, the interpretation of the significance of biphasic NH 4 Cl-induced ⌬pH i for RhCG function is not unequivocal. This ⌬pH i is consistent with the influx of both NH 3 and NH 4 ϩ but raises the question of whether the transport of NH 3 and NH 4 ϩ is stoichiometrically coupled. The biphasic ⌬pH i suggests that RhCG-mediated ⌽ NH 3 and ⌽ NH 4 both vary during NH 4 Cl exposure. This favors the hypothesis of uncoupled fluxes. Associated but uncoupled transport systems have been reported in the literature. For example, glutamate transporters are associated with thermodynamically uncoupled Cl Ϫ channels (27), and glutamine transporter SN1 is associated with a non-stoichiometrically coupled proton current (28). However, the observed biphasic ⌬pH i does not definitively rule out stoichiometric coupling of ⌽ NH 3 and ⌽ NH 4 . It could be that RhCG mediates a bidirectional NH 3 flux, as reported for AmtB protein (8). In that case, during NH 4 Cl exposure, the initial cell alkalinization results from inward fluxes of both ⌽ NH 3 and ⌽ NH 4 (the very partial dissociation of NH 4 ϩ only partly tempers the effect on pH i of the NH 3 protonation, thus cell alkalinization). If NH 3 reaches transmembrane equilibrium before NH 4 ϩ , a proton shuttle would be induced (29). If RhCG mediates bidirectional NH 3 flux (as reported for AmtB protein (8)), a reduced NH 3 net influx will occur. Because the NH 4 ϩ net influx is higher than NH 3 net influx, the cell acidifies. Additional studies are needed to determine whether RhCG-mediated NH 3 and NH 4 ϩ fluxes are stoichiometrically coupled.
In summary, our study shows that, when expressed in X. laevis oocytes, RhCG induces ammonium transport by a novel mechanism: NH 4 ϩ transport is enhanced by a mechanism depending on NH 3 . Moreover, our results provide evidence that human Rh proteins may not all share a unique functional role: in RhCG-expressing oocytes, NH 3 is involved in electrogenic NH 4 ϩ transport, whereas in RhAG-expressing oocytes, an NH 3independent, electroneutral NH 4 ϩ /H ϩ antiporter was reported (12). Because our results demonstrate enhanced NH 3 transmembrane transport in RhCG-expressing oocytes, RhCG may represent a facilitating pathway for transmembrane NH 3 diffusion. Other recent findings support the role of transmembrane proteins in gas transport (30 -32), despite the widely admitted theory of free gas diffusion across biological membranes. When expressed in X. laevis oocytes, the human protein RhCG mediates a highly specific ammonium transport with high substrate affinity but with complex behavior meriting further investigation to determine the mechanism of RhCG effects. RhCG function in the kidney remains to be investigated.