Abstract
We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH +4 ) under open-circuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH3 permeability was evident from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH3, AQP8, AQP9, AQP3, and TIP2;1 supported inwards currents carried by NH +4 . This conductivity increased as a sigmoid function of external [NH3]: for AQP8 at a bath pH (pHe) of 6.5, the conductance was abolished, at pHe 7.4 it was half maximal and at pHe 7.8 it saturated. NH +4 influx was associated with oocyte swelling. In comparison, native oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH3. These aquaporins could support NH +4 transport and have physiological implications for liver and kidney function.
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References
Ackerman MJ, Wickman KD, Clapham DE (1994) Hypotonicity activates a native chloride current in Xenopus oocytes. J Gen Physiol 103:153–179
Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S (2002) Aquaporin water channels—from atomic structure to clinical medicine. J Physiol (Lond) 542:3–16
Bakouh N, Benjelloun F, Hulin P, Brouillard F, Edelman A, Chèrif-Zahar B, Planelles G (2004) NH3 is involved in the NH +4 transport induced by the functional expression of the human Rh C glycoprotein. J Biol Biochem 279:15975–15983
Boldt M, Burckhardt G, Burckhardt BC (2003) NH +4 conductance in Xenopus laevis oocytes. III. Effect of NH3. Pflugers Arch 446:652–657
Burckhardt B-C, Frömter E (1992) Pathways of NH3/NH +4 permeation across Xenopus laevis oocyte cell membrane. Pflugers Arch 420:83–86
Burckhardt B-C, Burckhardt G (1997) NH +4 conductance in Xenopus laevis oocytes. Pflugers Arch 434:306–312
Calamita G, Mazzone A, Bizzoca A, Cavalier A, Cassano G, Thomas D, Svelto M (2001) Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract. Eur J Cell Biol 80:711–719
Carbrey JM, Gorelick-Feldman DA, Kozono D, Praetorius J, Nielsen S, Agre P (2003) Aquaglyceroporin AQP9: solute permeation and metabolic control of expression in liver. Proc Natl Acad Sci USA 100:2945–2950
Cougnon M, Bouyer P, Hulin P, Anagnostopoulos T, Planelles G (1996) Further investigation of ionic diffusive properties and of NH +4 pathways in Xenopus laevis oocyte cell membrane. Pflugers Arch 431:658–667
Elkjær M-L, Nejsum LN, Gresz V, Kwon T-H, Jensen UB, Frøkiær J, Nielsen S (2001) Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways. Am J Physiol 281:F1047–F1057
Engel A, Stahlberg H (2002) Aquaglyceroporins: channel proteins with a conserved core, multiple functions, and variable surfaces. Int Rev Cytol 215:75–104
Ferri D, Mazzone A, Liquori GE, Cassano G, Svelto M, Calamita G (2003) Ontogeny, distribution, and possible functional implications of an unusual aquaporin, AQP8, in mouse liver. Hepatology 38:947–957
Finkelstein A (1987) Water movement through lipid bilayers, pores and plasma membranes. Wiley-Interscience, New York
Garcia F, Kierbel A, Larocca MC, Gradilone SA, Splinter P, LaRusso NF, Marinelli RA (2001) The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP. J Biol Chem 276:12147–12152
Häussinger D (1996) Physiological functions of the liver. In: Greger R, Windhorst U (eds) Comprehensive human physiology, Vol. 2. Springer, Berlin Heidelberg New York, pp 1369–1391
Häussinger D (1996) Zonal metabolism in the liver. In: Greger R, Windhorst U (eds) Comprehensive human physiology, Vol 2. Springer, Berlin Heidelberg New York, pp 1393–1402
Hill EA (1994) Osmotic flow in membrane pores of molecular size. J Membr Biol 137:197–203
Holm LM, Klaerke DA, Zeuthen T (2004) Aquaporin 6 is permeable to glycerol and urea. Pflügers Arch 448:181–186
Huebert RC, Splinter PL, Garcia F, Marinelli RA, LaRusso NF (2002) Expression and localization of aquaporin water channels in rat hepatocytes. J Biol Chem 277:22710–22717
Ishibashi K, Kuwahara M, Gu Y, Tanaka Y, Marumo F, Sasaki S (1998) Cloning and functional expression of a new aquaporin (AQP9) abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol. Biochem Biophys Res Commun 244:268–274
Jahn TP, Møller ALB, Zeuthen T, Holm LM, Klaerke DA, Mohsin B, Kühlbrandt W, Schjoerring JK (2004) Aquaporin homologues in plants and mammals transport ammonia. FEBS Lett 574:31–36
Kedem O, Katchalsky A (1961) A physical interpretation of the phenomenological coefficients of membrane permeability. J Gen Physiol 45:143–179
Khademi S, O’Connell III J, Remis J, Robles-Colmenares Y, Miercke LJW, Stroud RM (2004) Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305:1587–1594
Knepper MA, Packer R, Good DW (1989) Ammonium transport in the kidney. Physiol Rev 69:179–249
Koyama Y, Yamamoto T, Kondo D, Funaki H, Yaoita E, Kawasaki K, Sato N, Hatekeyama K, Kihara I (1997) Molecular cloning of a new aquaporin from rat pancreas and liver. J Biol Chem 272:30329–30333
Ludewig U (2004) Electroneutral ammonium transport by basolateral rhesus B glycoprotein. J Physiol (Lond) 559:751–759
Ma T, Song Y, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS (2000) Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels. Proc Natl Acad Sci USA 97:4386–4391
Ma T, Yang B, Verkman AS (1997) Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver and heart. Biochem Biophys Res Commun 240:324–328
Meinild A-K, Klaerke DA, Loo DDF, Wright EM, Zeuthen T (1998) The human Na+ /glucose cotransporter is a molecular water pump. J Physiol (Lond) 508:15–21
Meinild A-K, Klaerke DA, Zeuthen T (1998) Bidirectional water fluxes and specificity for small hydrophilic molecules in aquaporins 0 to 5. J Biol Chem 273:32446–32451
Murata K, Mitsouka K, Hirai T, Walz T, Agre P, Heymann JB, Engel A, Fujiyoshi Y (2000) Structural determinants of water permeation through aquaporin-1. Nature 407:599–605
Nakhoul NL, Hamm LL (2004) Non-erythroid Rh glycoproteins: a putative new family of mammalian ammonium transporters. Pflugers Arch 447:807–812
Nakhoul NL, Hering-Smith KS, Abdulnour-Nakhoul SM, Hamm LL (2001) Transport of NH3/NH +4 in oocytes expressing aquaporin-1. Am J Physiol 281:F255–F263
Nicholls DG, Ferguson SJ (1995) Bioenergetics 2 edn. Academic Press, London
Nielsen S, Frøkier J, Marples D, Kwon T-H, Agre P, Knepper MA (2002) Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82:205–244
Portincasa P, Moschetta A, Mazzone A, Palasciano G, Svelto M, Calamita G (2003) Water handling and aquaporins in bile formation: recent advances and research trends. J Hepatol 39:864–874
Preston GM, Carroll TP, Guggino WB, Agre P (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256:385–389
Sasaki S, Ishibashi K, Marumo F (1998) Aquaporin-2 and -3: representatives of two subgroups of the aquaporin family colocalized in the kidney collecting duct. Annu Rev Physiol 60:199–220
Sasaki S, Ishibashi K, Nagai T, Marumo F (1992) Regulation mechanisms of intracellular pH of Xenopus laevis oocyte. Biochim Biophys Acta 1137:45–51
Tsukaguchi H, Shayakul C, Berfer UV, Mackenzie B, Devidas S, Guggino WB, VanHoek AN, Hediger MA (1998) Molecular characterization of a broad selectivity neutral solute channel. J Biol Biochem 273:24737–24743
Tsukaguchi H, Weremowicz S, Morton CC, Hediger MA (1999) Functional and molecular characterization of the human neutral solute channel aquaporin-9. Am J Physiol 277:F685–F696
Vaughan-Jones RD, Peercy BE, Keener JP, Spitzer KW (2002) Intrinsic H+ ion mobility in the rabbit ventricular myocyte. J Physiol (Lond) 541:139–158
Verkman AS, Yang B, Song Y, Manley GT, Ma T (2000) Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice. Exp Physiol 85S:233S–241S
Zampighi GA, Kreman M, Boorer KJ, Loo DDF, Bezanilla F, Chandy G, Hall JE, Wright EM (1995) A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes. J Membr Biol 148:65–78
Zeuthen T (1980) How to make and use double-barreled ion-selective microelectrodes. Curr Top Membr Trans 13:31–47
Zeuthen T, Hamann S, la Cour M (1996) Cotransport of H+, lactate and H2O by membrane proteins in retinal pigment epithelium of bullfrog. J Physiol (Lond) 497:3–17
Zeuthen T, Klaerke DA (1999) Transport of water and glycerol in aquaporin 3 is gated by H+. J Biol Chem 274:21631–21636
Zeuthen T, Meinild A-K, Klaerke DA, Loo DDF, Wright EM, Belhage B, Litman T (1997) Water transport by the Na+ /glucose cotransporter under isotonic conditions. Biol Cell 89:307–312
Zeuthen T, Zeuthen E, Klaerke DA (2002) Mobility of ions, sugar, and water in the cytoplasm of Xenopus oocytes expressing Na+-coupled sugar transporters (SGLT1). J Physiol (Lond) 542:71–87
Acknowledgements
The authors are grateful for expert technical assistance from B. Lynderup, T. Soland and S. Christoffersen and for valuable discussions with Dr. E. Beitz. The study was supported by the Nordic Centre of Excellence Programme in Molecular Medicine, The Danish Research Council Veluxfonden, Øjenforeningen, Alice and Jørgen Rasmussens mindelegat and the Lundbeck Foundation to T.Z., SJVF (23-03-0103) to T.P.J and the Centro di Eccellenza di Genomica in campo Biomedico ed Agrario and Fondo per gli Investimenti della Ricerca di Base (RBAU01RANB).
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Appendix
In aqueous solutions of NH4Cl, NH +4 is present together with its conjugate base NH3. Under equilibrium conditions, \({\rm NH}_{4}^{+} = {\rm NH}_{3} \times 10\,^{pK-pH}\) with pK=9.25. Thus, for the external solution:
Consequently, if the relative rate of influx of NH3 is larger than that of NH +4 , the external solution acidifies. With \({\rm dNH}_{3}/{\text{d}}t = - P_{NH3} \times {\rm NH}_{3}\) and \({\rm dNH}_{4}^{+}/{\text{d}}t = - P_{NH4}^{+} \times {\rm NH}_{4}^{+}, \) Eq. 4 becomes
In other words, if PNH3 is larger than PNH4+ the external solution will acidify. Note that PNH4+ incorporates electrical terms.
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Holm, L.M., Jahn, T.P., Møller, A.L.B. et al. NH3 and NH +4 permeability in aquaporin-expressing Xenopus oocytes. Pflugers Arch - Eur J Physiol 450, 415–428 (2005). https://doi.org/10.1007/s00424-005-1399-1
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DOI: https://doi.org/10.1007/s00424-005-1399-1