NH3 Is Involved in the NH 4 + Transport Induced by the Functional Expression of the Human Rh C Glycoprotein
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m308528200
ISSN1083-351X
AutoresNaziha Bakouh, Fatine Benjelloun, Philippe Hulin, Franck Brouillard, Aleksander Edelman, Baya Chérif‐Zahar, Gabrielle Planelles,
Tópico(s)Metabolism and Genetic Disorders
ResumoRenal 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 μm 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. 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 μm 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. 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 (1Liu Z. Peng J. Mo R. Hui C. Huang C.H. J. Biol. Chem. 2001; 276: 1424-1433Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and that RhCG is expressed mainly in the testis and kidney (2Liu Z. Chen Y. Mo R. Hui C. Cheng J.F. Mohandas N. Huang C.H. J. Biol. Chem. 2000; 275: 25641-25651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 3Marini A.M. Matassi G. Raynal V. André B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (300) Google Scholar). Rh proteins share homologies with the Mep/Amt family from yeasts, bacteria, and plants (4Marini A.M. Urrestarazu A. Beauwens R. Andre B. Trends Biochem. Sci. 1997; 22: 460-461Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 5Matassi G. Chérif-Zahar B. Raynal V. Rouger P. Cartron J.P. Genomics. 1998; 47: 286-293Crossref PubMed Scopus (39) Google Scholar). Despite numerous studies implicating Mep/Amt proteins in ammonium transport (6Ninnemann O. Jauniaux J.C. Frommer W.B. EMBO J. 1994; 13: 3464-3471Crossref PubMed Scopus (330) Google Scholar, 7Marini A.M. Soussi-Boudekou S. Vissers S. André B. Mol. Cell. Biol. 1997; 17: 4282-4293Crossref PubMed Scopus (458) Google Scholar), 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 (8Soupene E. Lee H. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3926-3931Crossref PubMed Scopus (100) Google Scholar) and, on the other hand, that LeAMT1;1 acts as an NH4+ uniport after its functional expression in Xenopus laevis oocytes (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Consistent with the involvement of Rh proteins in transmembrane ammonium transport, yeasts deficient in endogenous ammonium transport system (ΔMep Saccharomyces cerevisiae) are enabled to grow in a low ammonium-containing medium when transformed by RhAG or by RhCG (3Marini A.M. Matassi G. Raynal V. André B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (300) Google Scholar). Based on their finding that Mep and Amt are NH3 channels, Soupene et al. (10Soupene E. Ramirez R.M. Kustu S. Mol. Cell. Biol. 2001; 21: 5733-5741Crossref PubMed Scopus (76) Google Scholar, 11Soupene E. King N. Feild E. Liu P. Niyogi K.K. Huang C.H. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7769-7773Crossref PubMed Scopus (116) Google Scholar) raised the hypothesis that Rh proteins are involved in gas transport rather than in ionic transport. However, Westhoff et al. (12Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) concluded that RhAG mediates an electroneutral ionic exchange of NH4+ 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 ammonium 1In the text, "ammonium" is used when ammonia and ammonium ions are not discriminated. The chemical symbols for ammonia (NH3) and ammonium ions (NH4+) are used to distinguish the two forms of ammonium. (NH4+ 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 NH4+-related current. However, the amplitude of IAM was strongly sensitive to changes in extracellular pH (pHo) of ±0.5 pH unit (pH U), 2The 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. an experimental maneuver that only slightly changes [NH4+] 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 NH4+/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 NH4+ influx into the cell. These results are consistent with RhCG-induced NH3 and NH4+ transport. 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. 13Bissig K.D. La Fontaine S. Mercer J.F. Solioz M. Biol. Chem. 2001; 382: 711-714Crossref PubMed Scopus (8) Google Scholar). 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 (3Marini A.M. Matassi G. Raynal V. André B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (300) Google Scholar) 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 (14Cougnon M. Planelles G. Crowson M.S. Shull G.E. Rossier B.C. Jaisser F. J. Biol. Chem. 1996; 271: 7277-7280Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). 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 (15Cougnon M. Bouyer P. Planelles G. Jaisser F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6516-6520Crossref PubMed Scopus (79) Google Scholar, 16Prie D. Huart V. Bakouh N. Planelles G. Dellis O. Gérard B. Hulin P. Benque-Blanchet F. Silve C. Grandchamp B. Friedlander G. N. Engl. J. Med. 2002; 347: 983-991Crossref PubMed Scopus (289) Google Scholar). 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 [NH4+] in ammonium-containing solutions, the pKa was taken as 9.25. As reported by other groups (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Burckhardt B.C. Thelen P. Pfluegers Arch. Eur. J. Physiol. 1995; 429: 306-312Crossref PubMed Scopus (33) Google Scholar), 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 (17Burckhardt B.C. Thelen P. Pfluegers Arch. Eur. J. Physiol. 1995; 429: 306-312Crossref PubMed Scopus (33) Google Scholar). This is likely related to the multiple endogenous cationic conductances that are activated by NH4Cl in native X. laevis oocyte (18Cougnon M. Bouyer P. Hulin P. Anagnostopoulos T. Planelles G. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 658-667Crossref PubMed Scopus (43) Google Scholar). 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 (19Anagnostopoulos T. Planelles G. J. Physiol. (Lond.). 1987; 393: 73-89Crossref Scopus (25) Google Scholar). 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). 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 (3Marini A.M. Matassi G. Raynal V. André B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (300) Google Scholar). 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. 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, NH4+) 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. 20Rettinger J. Pfluegers Arch. Eur. J. Physiol. 1999; 437: 917-924Crossref PubMed Scopus (12) Google Scholar) 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 NH4+ or of H+ ions (18Cougnon M. Bouyer P. Hulin P. Anagnostopoulos T. Planelles G. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 658-667Crossref PubMed Scopus (43) Google Scholar). 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, ΔpHi = 0.02 ± 0.01; p = 0.3; n = 4), we next determined the specificity of the currents to NH4+ compared with other cationic species. 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 NH4+ 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 NH4+ 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 (3Marini A.M. Matassi G. Raynal V. André B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (300) Google Scholar). 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+] (18Cougnon M. Bouyer P. Hulin P. Anagnostopoulos T. Planelles G. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 658-667Crossref PubMed Scopus (43) Google Scholar), 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 (11Soupene E. King N. Feild E. Liu P. Niyogi K.K. Huang C.H. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7769-7773Crossref PubMed Scopus (116) Google Scholar, 12Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). RhAG, after functional expression in X. laevis oocytes, was reported to mediate an electrically silent NH4+/H+ exchange (12Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). In that study, the authors proposed that this electroneutral ionic transport mediated by RhAG would not change pHi, despite the RhAG-induced NH4+ influx into the cell (12Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Another group speculated that human Rh proteins mediate CO2 gas diffusion (11Soupene E. King N. Feild E. Liu P. Niyogi K.K. Huang C.H. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7769-7773Crossref PubMed Scopus (116) Google Scholar). 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 (11Soupene E. King N. Feild E. Liu P. Niyogi K.K. Huang C.H. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7769-7773Crossref PubMed Scopus (116) Google Scholar). 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 NH4+ and K+ in RhCG-expressing oocytes, as well as the observation that Na+,K+, and choline+ do not mimic the effect of NH4+ on membrane current, agrees with results reported in X. laevis oocytes expressing LeAMT1;1, an Amt protein from tomato root hair (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). 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 NH4+ uniport (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The NH4+ currents induced by LeAMT1;1 expression were pHo-independent (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). 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 NH4+ concentration ([NH4+] 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 [NH4+] (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]. The above-mentioned results show a strong effect of changing pH/NH3 on NH4+-induced current in RhCG-expressing oocytes. This is at variance with results reported for LeAMT1;1-expressing oocytes (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). In that study, the pHo independence of IAM was invoked as a strong argument to conclude that LeAMT1;1 mediates an NH4+ uniport, independent of NH3 (9Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Our results also argue against a putative NH4+-H+ co-transport mediating both NH4+ 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 NH4+, 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 [NH4+] 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 [NH4+] because NH4+ 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 [NH4+]) 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 (12Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). This finding was taken as an argument against the transport by RhAG of NH3 or of the neutral form of methylammonium (12Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Interestingly, in RhCG-expressing oocytes, the apparent NH3 affinity (≈8 μm) seems to be pHo-independent. In another experimental series, RhCG-expressing oocytes were exposed to increasing [NH3] at constant [NH4+] (pHo was changing). This protocol was carried out at constant [NH4+] ≈ 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. In a complementary protocol, RhCG-expressing oocytes were exposed to increasing [NH4+] 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 [NH4+] 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 NH4+ was at a saturating concentration. To discriminate between these possibilities, RhCG-expressing oocytes were again exposed to increasing [NH4+] at constant [NH3], but the previous protocol was changed in two ways. First, to obtain solutions containing less [NH4+], 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 [NH4+] ≈ 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 NH4+ transport system dependent on NH3. Finally, to check whether pHo per se affects RhCG function, we measured the current induced by [NH3] and [NH4+] 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 (17Burckhardt B.C. Thelen P. Pfluegers Arch. Eur. J. Physiol. 1995; 429: 306-312Crossref PubMed Scopus (33) Google Scholar). 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 NH4+-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 NH4+, decreasing it at alkaline pHo and increasing it at acidic pHo. The results also agree with the dependence of IAM on the concentrations of both the charged (nam
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