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J. Biol. Chem., Vol. 279, Issue 16, 15975-15983, April 16, 2004
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NH3 Is Involved in the 
Transport Induced by the Functional Expression of the Human Rh C Glycoprotein*
From the INSERM U 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France
Received for publication, August 4, 2003 , and in revised form, January 28, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Renal ammonium (NH3 +
) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow transmembrane translocation without high specificity , 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 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 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 µM NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH3 and transport. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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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 NH3 diffusion across the cytoplasmic membrane of Salmonella typhimurium (8) and, on the other hand, that LeAMT1;1 acts as an
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 NH3 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 for H+ 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 ammonium1 ( and/or NH3) transport. Exposure of voltage-clamped RhCG-expressing cells to submillimolar concentrations of NH4Cl ([NH4Cl]) induced inward currents (IAM). The amplitude of IAM increased with [NH4Cl] and membrane hyperpolarization, consistent with an -related current. However, the amplitude of IAM was strongly sensitive to changes in extracellular pH (pHo) of ±0.5 pH unit (pH U),2 an experimental maneuver that only slightly changes [] but substantially affects [NH3]. At alkaline but not physiological pHo, the enhancement of methylammonium-induced current by micromolar [NH4Cl] further supports the requirement of the neutral form for /methylammonium transport in RhCG-expressing oocytes. Exposure to 500 µM NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and influx into the cell. These results are consistent with RhCG-induced NH3 and transport.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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cRNA Synthesis and Expression in X. laevis Oocytes—To routinely control RhCG expression in X. laevis oocytes, we constructed an N-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 ± 40 nm; emission filter, 505 nm). RhCG cDNA was amplified by PCR from the pRS426-RhCG construct (3) using 5'-CTGCAGCATGGCCTGGAACACCAACCT-3' and 5'-CTCCTCACCTGCCCTGGGAGCCTAGGG-3' 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 = –50 mV. Oocytes were superfused by a control Ringer solution adapted for amphibia (containing 96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 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 µM NH4Cl-containing solution at pHo 7.5. Solutions at pHo = 7.0 or 8.0 were also buffered with Hepes/NaOH. Solutions at pHo = 8.5 were buffered with TAPS/NaOH (switching from Hepes to TAPS buffer, at identical pHo, had no effect). In Na+-free experiments, NaCl was replaced by an equimolar concentration of choline chloride, and pH was adjusted using Trizma (Tris base). To calculate [NH3] and [] in ammonium-containing solutions, the pKa was taken as 9.25.
As reported by other groups (9, 17), we noticed that in control oocytes, even at low millimolar concentrations, NH4Cl (and methylammonium) may induce an inward endogenous current that increases at alkaline pHo (17). This is likely related to the multiple endogenous cationic conductances that are activated by NH4Cl in native X. laevis oocyte (18). In the present study, such endogenous currents were detected in H2O-injected oocytes upon exposure to [NH4Cl] 
3 mM, [NH4Cl] 1.5 mM, and [NH4Cl] 500 µM at pHo = 7.0, 7.5, and 8.0, respectively. At these low [NH4Cl], the endogenous current amplitude (Iendog) varied from one batch of oocytes to another. Thus, when applying corrections, the NH4Cl-induced response in control cells was subtracted from the NH4Cl-induced response measured in RhCG-expressing oocytes from the same batch. However, because Iendog in oocytes may also vary within the same batch of oocytes, we avoided as far as possible the use of NH4Cl concentrations high enough to induce an endogenous response, except when necessary for further characterization of RhCG functional properties.
Intracellular pH Measurements—Intracellular pH (pHi) and membrane potential (Vm) were simultaneously measured using double-barreled pH-sensitive microelectrodes (filled with the Fluka H+ 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 pHo. In our experiments, S was 55–59 mV/pH U. pHi was calculated using the relationship pHi = pHo – (VH – Vm)/S, where VH is the proton electrochemical potential difference across the cell membrane.
Except where stated, results were expressed as means ± S.E. (with n = number of oocytes), and the significance of the results was assessed by paired or unpaired Student t test (p < 0.05 was considered significant).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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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 [NH4Cl] (100, 250, 500, and 1000 µM; pHo = 7.5) was investigated in voltage-clamped cells. In control oocytes (n = 21), this experimental maneuver was without effect, except occasionally for [NH4Cl] = 1000 µM (which may induce a small endogenous inward current, up to –5 nA). In RhCG-expressing oocytes, this experimental maneuver induced increasing inward currents, as shown in Fig. 1. In this series, we also measured pHi. Resting pHi was not modified by RhCG expression (pHi = 7.44 ± 0.02 (n = 23) in RhCG-expressing oocytes versus 7.39 ± 0.03 (n = 18) in control oocytes; p = 0.2). This rules out a pHi change as the explanation for IAM 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 IAM, 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.
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, [NH4Cl] = 100 µM; 
, [NH4Cl] = 250 µM; 
, [NH4Cl] = 500 µM; 
, [NH4Cl] = 1000 µM. A, tracing obtained from an RhCG-expressing oocyte (injected with 10 ng of cRNA). [NH4Cl] induced inward currents, increasing with increasing [NH4Cl]. B, tracing obtained from a control oocyte (injected with H2O). No detectable NH4Cl-induced current is observed, except in the presence of [NH4Cl] = 1000 µM (the slight inward current observed was not frequently detected in control oocytes). Inset, effect of increasing cRNA on NH4Cl-induced current (I, indicated on the ordinate as means ± S.E.) in RhCG-expressing oocytes injected with increasing [RNA], as indicated below the abscissa.Specificity of NH4Cl-induced Currents—The voltage dependence of IAM in RhCG-expressing oocytes was investigated by applying a [NH4Cl] = 500 µM pulse at various holding potential values. The resulting current-voltage relationship (Fig. 2) shows that IAM increased with hyperpolarization (inside negative), consistent with the enhancement of net entry of positive charges (namely,
) 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+ conductance induced by depolarization; Ref. 20) gives a reversal potential (Erev) near 0 mV. This Erev value may correspond to the combined equilibrium potential of the main cationic species (Na+ + K+) of or of H+ ions (18). Because a major involvement of H+ ions was not supported by the pHi stability of RhCC-expressing oocytes upon pHo change (from pHo 7.5 to 8.5, pHi = 0.02 ± 0.01; p = 0.3; n = 4), we next determined the specificity of the currents to compared with other cationic species.
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First, voltage-clamped oocytes (Vc = –50 mV) were exposed pairwise to a Ringer solution supplemented with 500 µM NH4Cl or 500 µM choline chloride or NaCl. Neither NaCl nor choline chloride induced a current in RhCG-expressing oocytes or control cells (data not shown), whereas IAM was –13.2 ± 1.1 nA in RhCG-expressing oocytes (n = 8) but was not detectable in H2O-injected cells (IAM = 0.1 ± 0.2; n = 4). The non-involvement of Na+ ions in RhCG function was further confirmed by measuring in a paired fashion that the current induced by 500 µM NH4Cl added to a plain Ringer solution or to a Na+-free solution was the same (n = 10; p = 0.3). This also confirms that RhCG does not mediate a nonselective cationic pathway, in which case IAM would be expected to increase under Na+-free conditions.
Second, the discrimination between K+ and in RhCG-expressing oocytes was investigated. To this end, the effect of adding 500 µM KCl to the Ringer solution was checked. This maneuver did not induce an inward current in RhCG-expressing oocytes, consistent with high selectivity for over K+. This finding is in agreement with studies in S. cerevisiae showing that RhCG did not complement the growth defect of yeast deficient in K+ transport (3). In fact, in this series, we observed a slight KCl-induced outward current in all oocytes (+2.1 ± 0.6 nA in RhCG-expressing oocytes and +0.8 ± 0.7 nA in H2O-injected oocytes; n = 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+] (18), which was increased by 25% in our experiments. The non-involvement of K+ in RhCG function was confirmed by measuring in a paired fashion that the current induced by 500 µM NH4Cl added in a plain Ringer solution or in a K+-free medium was the same (n = 3; p = 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 /H+ exchange (12). In that study, the authors proposed that this electroneutral ionic transport mediated by RhAG would not change pHi, despite the RhAG-induced influx into the cell (12). Another group speculated that human Rh proteins mediate CO2 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-CO2 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 CO2 diffusion by RhCG. With regard to NH3, which is also a gas, RhCG-mediated transport of this uncharged species cannot account for the observed IAM, at least not in a simple manner. However, the rheogenicity of the ammonium-induced response does not exclude the possibility that NH3 is transported together with an ion or that NH3 stimulates an electrogenic ionic transport in RhCG-expressing oocytes. In the experiments reported above, the discrimination observed between and K+ in RhCG-expressing oocytes, as well as the observation that Na+,K+, and choline+ do not mimic the effect of 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 NH4Cl-induced Currents— When it is expressed in X. laevis oocytes, the Amt protein LeAMT1;1 mediates an uniport (9). The currents induced by LeAMT1;1 expression were pHo-independent (9). To determine whether LeAMT1;1 and RhCG have similar functional properties, we looked for the effect of changing pHo on IAM in voltage-clamped oocytes.
We measured the current induced by a given NH4Cl concentration by exposing the cells in a paired fashion to 500 µM NH4Cl at pHo 7.5 and then at pHo 7.0 or 8.0. In RhCG-expressing oocytes, at pHo = 7.0, IAM was barely measurable (IAM = –1.2 ± 0.5 nA at pHo = 7.0 versus –13.2 ± 1.7 nA at pHo = 7.5; n = 10; p < 0.05), whereas it was greatly enhanced at pHo = 8.0 (IAM =–28.2 ± 2.8 nA at pHo = 8.0 versus –8.6 ± 0.6 at pHo = 7.5; n = 9; p < 0.05). Such large changes in IAM were surprising because at constant [NH4Cl] = 500 µM, changing pHo by ± 0.5 pH U only marginally affects concentration ([] decreases by 
25 µM from pHo 7.0 to 8.0). However, this change of pHo by ± 0.5 pH U causes a 3-fold change in [NH3]. To confirm that a pHo/NH3 change directly affects IAM, we compared the current induced by [NH4Cl] = 500 µM at pHo = 7.5 with the currents induced by NH4Cl-containing solutions with either the same [NH3] or the same [] (see Fig. 3). These solutions were buffered to pH 7.0 (Fig. 3A) or pH 8.0 (Fig. 3B). In both series, IAM was the same during exposure of RhCG-expressing oocytes to solutions with different pHo but containing identical [NH3].
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The above-mentioned results show a strong effect of changing pH/NH3 on
-induced current in RhCG-expressing oocytes. This is at variance with results reported for LeAMT1;1-expressing oocytes (9). In that study, the pHo independence of IAM was invoked as a strong argument to conclude that LeAMT1;1 mediates an uniport, independent of NH3 (9). Our results also argue against a putative -H+ co-transport mediating both and H+ influx into the cell. Such a transport system would induce an IAM inward current, but alkaline external pH should reduce IAM (conversely, acidic pH should increase IAM), whereas we observed the opposite. These results suggest that NH3 or H+ ions are directly involved in RhCG function.
Substrate Dependence of IAM—Discrimination between H+ and NH3 effects is not obvious because of the equilibrium reaction between , NH3, and H+. 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, IAM was measured while keeping pHo, [NH3], or [] constant while varying the other two.
First, RhCG-expressing oocytes were exposed to increasing [NH4Cl] at a constant pHo of 7.5 (Fig. 4A) or 7.0 (Fig. 4B). This protocol gives the dependence of IAM on [NH4Cl]. This represents, to a first approximation, the IAM dependence on [] because concentration is so much higher than that of NH3 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 []) appears to be pHo-dependent. This observation is at variance with results reported for the function of RhAG because changes in pHo 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 NH3 or of the neutral form of methylammonium (12). Interestingly, in RhCG-expressing oocytes, the apparent NH3 affinity (8 µM) seems to be pHo-independent.
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In another experimental series, RhCG-expressing oocytes were exposed to increasing [NH3] at constant [
] (pHo was changing). This protocol was carried out at constant [] 491 µM (Fig. 5A) or 246 µM (Fig. 5B). Results confirmed that half-maximal IAM was reached for [NH3] 8 µM (corresponding to different pHo) as in the previous series (Fig. 4). This finding suggests NH3 transport by RhCG and/or its direct involvement in IAM.
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In a complementary protocol, RhCG-expressing oocytes were exposed to increasing [
] at constant [NH3] (in the same range of pHo values as in the previous protocol, i.e. from pHo 7.0 to 8.0 by increments of 0.2 pH U). This experimental series was performed with [NH3] constant at 8.73 or 4.36 µM. Analysis (by analysis of variance) of the results (data not shown) obtained in n = 5–7 oocytes showed that IAM was constant for a given [NH3], despite the 10-fold change imposed in [] between pH 7.0 and 8.0. These results agree with results from Fig. 3 and suggest either that [NH3] acts as an "on-off switch" for IAM or that was at a saturating concentration. To discriminate between these possibilities, RhCG-expressing oocytes were again exposed to increasing [] at constant [NH3], but the previous protocol was changed in two ways. First, to obtain solutions containing less [], pHo was raised to 8.6 or 8.8. To obtain a better resolution of the changes in IAM, [NH3] = 13.09 µM rather than [NH3] = 4.36 µM was used. Results shown in Fig. 5, C and D, show that half-maximal IAM was reached for [] 140 and 120 µM for [NH3] = 13.09 and 8.73 µM, respectively. These results show that [NH3] does not act as an on-off switch for IAM. They are consistent with the expression in RhCG-expressing oocytes of a high affinity transport system dependent on NH3.
Finally, to check whether pHo per se affects RhCG function, we measured the current induced by [NH3] and [] above their respective saturating concentrations as determined from Figs. 4 and 5. To this end, the current induced by [NH4Cl] = 5 mM was measured at various pHo values. In H2O-injected oocytes, this maneuver induced an endogenous current (Iendog) that increased with increasing pHo, in agreement with a previous report (17). In RhCG-expressing oocytes, this maneuver induced a total current, ITOT, due to Iendog + IAM. Because Iendog variability appears to be high (especially at alkaline pHo) not only from one batch of oocytes to another but also within the oocytes of a given batch, we did not calculate IAM from ITOT – Iendog. Fig. 6 shows that [NH4Cl] = 5 mM induced significantly higher ammonium-induced current in RhCG-expressing oocytes than in H2O-injected oocytes. Results from Fig. 6 are also consistent with an increase of IAM when raising pHo and vice versa. This suggests that extracellular pH influences the maximal -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 pHo effect modified the value of Km for , decreasing it at alkaline pHo and increasing it at acidic pHo.
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) and RhCG-expressing oocytes (
) is indicated on the ordinate. pHo values are indicated on the abscissa. Results are expressed as mean ± S.E. (n = 11–18 control oocytes; n = 10–12 RhCG-expressing oocytes).The results also agree with the dependence of IAM on the concentrations of both the charged (namely,
) and uncharged (namely, NH3) forms of ammonium in RhCG-expressing oocytes. To better understand the role of NH3 in transport and RhCG function, we next investigated its effect on the transport of analogues by RhCG and the effect of NH4Cl exposure on intracellular pH.
Effect of Neutral Form on Current Induced by Analogues of NH+4—To clarify whether, in RhCG function, the neutral form activates an ionic current related to the charged form, we investigated the effect of methylamine (H3C-NH2/H3C- 
) 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, IMeA, at pHo = 7.5. Because H3C-NH2/H3C- has a higher pKa than NH3/ (10.65 versus 9.25) for identical total concentrations of NH4Cl or MeACl at pHo 7.5, methylammonium ions (H3C-) are in slightly higher concentration than (thus a lower H3C-NH2 concentration than NH3). However, 500 µM MeACl induced no current in either control or RhCG-expressing cells (IMeA = –0.4 ± 0.3 nA in RhCG-expressing oocytes (n = 17) and –0.2 ± 0.02 in control oocytes (n = 11)). Of note, in RhCG-expressing oocytes, a small IMeA was recorded when [MeACl] was increased to 750 (–3.0 ± 0.2 nA; n = 4) or 1000 µM (–3.6 ± 0.9 nA; n = 4), whereas no current was detectable in control cells (n = 3).
Second, we looked for a possible change in the current induced by 500 µM NH4Cl upon simultaneous exposure to NH4Cl and MeACl (500 µM each). Whereas this experimental maneuver was without effect in H2O-injected oocytes (n = 8), exposure of RhCG-expressing oocytes simultaneously to MeACl and NH4Cl induced a higher current than NH4Cl alone (IAM+MeA = –30.9 ± 3.1 versus IAM = –23.4 ± 3.8 nA; n = 4; p < 0.05). Increasing [MeACl] to 750 or 1000 µM in the presence of 500 µM NH4Cl led to a slight increase of IAM+MeA to –32.1 ± 3.0 and –33.7 ± 2.8 nA (n = 4), thus saturating. These results suggest that H3C- behaves as a low affinity substrate in RhCG function. Interestingly, our data show that IAM+MeA > IAM + IMeA, which may reflect a stimulation of /H3C- transport.
From these results, we suspected that in the above experiments, MeACl appeared to be a poor substitute substrate due to the very low concentration of H3C-NH2 (0.35 µM) at pHo 7.5. Thus, we measured the effect of 500 µM MeACl at pHo 8.5, an experimental condition in which H3C-NH2 was raised to 3.5 µM.IMeA was –5.6 ± 1.1 nA, a lower value than the paired IAM (–9.1 ± 0.8 nA; n = 11; p < 0.05) obtained with 500 µM NH4Cl/pHo 7.5 (i.e. [NH3] 8.5 µM). This result confirms that H3C- may substitute for and agrees with an effect of the noncharged form of the substrate on the current (reflecting the influx of the charged form of the substrate) amplitude.
Next, we searched for a stimulation of IMeA when providing a concentration of NH3 near the value determined for half-maximal IAM, while taking care to keep [] as low as possible. To this end, 50 µM NH4Cl was used at pHo = 8.5 (i.e. [NH3] 8 µM). As shown in Fig. 7, adding 50 µM NH4Cl to 500 µM MeACl increased the induced current by an amount similar to that obtained at pHo 7.5 in the presence of 500 µM NH4Cl (p = 0.6; n = 14). At pHo 7.5, the same maneuver was without effect (Fig. 7). This result was expected because at pHo 7.5, in a solution of 50 µM NH4Cl, the [NH3] is <1 µM, which is very low compared with the measured half-maximal concentration of 8 µM.
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Taken together, our results agree with NH3 involvement in
(H3C-)-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 [NH3] (12). In RhCG-expressing oocytes, our results suggest an electrogenic methylammonium influx activated by [NH3].
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 NH4Cl-induced intracellular Ca2+ increase (21). However, a similar explanation cannot account for our results because ammonium-induced activation of AE2 was observed in the presence of [NH4Cl] = 10–20 mM (21), an experimental condition very different from our conditions (stimulation of IMeA was observed using micromolar concentrations of NH3/). Moreover, in our experiments, IMeA 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, 22–24). 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 /H3C- is stimulated by the neutral form of these compounds in RhCG-expressing oocytes.
In light of these results suggesting that [NH3] stimulates IMeA, we further checked our previous conclusion that RhCG discriminates between and K+ (known to be competitive substrates on various transport systems) because the observed lack of K+-induced inward current in RhCG-expressing oocytes was obtained using NH3-free solutions. Thus, we looked for an induced current when adding 50 µM NH4Cl to a Ringer solution supplemented with 500 µM KCl at pHo = 8.5. This maneuver did not induce an inward current in RhCG-expressing oocytes (p = 0.5; n = 3). This further confirms that RhCG function is specifically related to ammonium (methylammonium) transport.
Effect of NH4Cl Exposure on Intracellular pH—To better determine whether NH3 is transported with , we measured the effect on pHi of exposing oocytes to 500 µM NH4Cl (pHo = 7.5). Control and RhCG-expressing oocytes exhibited the same resting pHi (pHi = 7.38 ± 0.02 (n = 9) versus 7.39 ± 0.01 (n = 9)), but exposure to NH4Cl induced a pHi change (biphasic change) only in RhCG-expressing oocytes, as shown in Fig. 8. In RhCG-expressing oocytes, the initial effect of NH4Cl exposure was a slight transient alkalinization (pHi increased to 7.42 ± 0.01, which was significantly different from resting pHi, p < 0.05), which was not detected in H2O-injected oocytes (pHi was stable (7.38 ± 0.02) and was not different from resting pHi, p = 0.3). During NH4Cl exposure, pHi significantly decreased to 7.34 ± 0.01 in RhCG-expressing oocytes but was unchanged in control oocytes (7.38 ± 0.03). Using a 20 mM NH4Cl-containing solution, we had previously reported that the oocyte membrane is only slightly permeable to NH3 compared with but that initial NH3 influx, 
NH3, into the cell is reflected by a small but detectable cell alkalinization preceding the cell acidification due to influx, NH (18, 26). In the present study, application of 500 µM NH4Cl had 4no effect on pHi in control oocytes. This indicates that this NH4Cl concentration is too low to induce any detectable NH3 or NH4 through the oocyte endogenous membrane pathways for ammonium. Thus, in the presence of 500 µM NH4Cl, the biphasic pHi change observed in RhCG-expressing oocytes reflects enhanced NH3 and NH4 as compared with control oocytes. One may argue that RhCG expression has simply enhanced endogenous NH3 and transport in the oocyte. However, the saturation of IAM as a function of [NH4Cl] (Fig. 4) does not agree with published characteristics of oocyte endogenous pathways (18), thus supporting our conclusion that RhCG expression induces a heterologous transport of both NH3 and .
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Nonetheless, the interpretation of the significance of biphasic NH4Cl-induced
pHi for RhCG function is not unequivocal. This pHi is consistent with the influx of both NH3 and but raises the question of whether the transport of NH3 and is stoichiometrically coupled. The biphasic pHi suggests that RhCG-mediated NH3 and NH4 both vary during NH4Cl 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– channels (27), and glutamine transporter SN1 is associated with a non-stoichiometrically coupled proton current (28). However, the observed biphasic pHi does not definitively rule out stoichiometric coupling of NH3 and NH4. It could be that RhCG mediates a bidirectional NH3 flux, as reported for AmtB protein (8). In that case, during NH4Cl exposure, the initial cell alkalinization results from inward fluxes of both NH3 and NH4 (the very partial dissociation of only partly tempers the effect on pHi of the NH3 protonation, thus cell alkalinization). If NH3 reaches transmembrane equilibrium before , a proton shuttle would be induced (29). If RhCG mediates bidirectional NH3 flux (as reported for AmtB protein (8)), a reduced NH3 net influx will occur. Because the net influx is higher than NH3 net influx, the cell acidifies. Additional studies are needed to determine whether RhCG-mediated NH3 and fluxes are stoichiometrically coupled.
In summary, our study shows that, when expressed in X. laevis oocytes, RhCG induces ammonium transport by a novel mechanism: transport is enhanced by a mechanism depending on NH3. Moreover, our results provide evidence that human Rh proteins may not all share a unique functional role: in RhCG-expressing oocytes, NH3 is involved in electrogenic transport, whereas in RhAG-expressing oocytes, an NH3-independent, electroneutral /H+ antiporter was reported (12). Because our results demonstrate enhanced NH3 transmembrane transport in RhCG-expressing oocytes, RhCG may represent a facilitating pathway for transmembrane NH3 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.
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FOOTNOTES |
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* This work was supported in part by INSERM and by Necker-Enfants Malades Faculty of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by fellowships from Vaincre la Mucoviscidose and Association Mucoviscidose, ABCF Proteine.
To whom correspondence should be addressed: INSERM U 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. Tel.: 33-1-40-61-56-20; Fax: 33-1-40-61-55-91; E-mail: planelle{at}necker.fr.
1 In the text, "ammonium" is used when ammonia and ammonium ions are not discriminated. The chemical symbols for ammonia (NH3) and ammonium ions () 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}-1-propanesulfonic acid; MeACl, methylamine chloride.
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ACKNOWLEDGMENTS |
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We thank Dr. S. R. Thomas for expert advice and acknowledge the excellent technical assistance of M. Blonde. G. P. is indebted to Dr. D. Hurvy for valuable discussions.
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REFERENCES |
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