Urea transporter B (UT-B) is a passive membrane channel that facilitates highly efficient permeation of urea. In red blood cells (RBC), while the major function of UT-B is to transport urea, it is assumed that this protein is able to conduct water. Here, we have revisited this last issue by studying RBCs and ghosts from human variants with defects of aquaporin 1 (AQP1) or UT-B. We found that UT-B's osmotic water unit permeability (pfunit) is similar to that of AQP1. The determination of diffusional permeability coefficient (Pd) allowed the calculation of the Pf/Pd ratio, which is consistent with a single-file water transport. Molecular dynamic simulations of water conduction through human UT-B confirmed the experimental finding. From these results, we propose an atomistic description of water–protein interactions involved in this permeation. Inside the UT-B pore, five water molecules were found to form a single-file and move rapidly along a channel by hydrogen bond exchange involving two critical threonines. We further show that the energy barrier for water located in the central region coincides with a water dipole reorientation, which can be related to the proton exclusion observed experimentally. In conclusion, our results indicate that UT-B should be considered as a new member of the water channel family.
References
[1]
Lucien N, Sidoux-Walter F, Roudier N, Ripoche P, Huet M, et al. (2002) Antigenic and functional properties of the human red blood cell urea transporter hUT-B1. J Biol Chem 277: 34101–34108.
[2]
Mathai JC, Mori S, Smith BL, Preston GM, Mohandas N, et al. (1996) Functional analysis of aquaporin-1 deficient red cells. The Colton-null phenotype. J Biol Chem 271: 1309–1313.
[3]
Macey RI (1984) Transport of water and urea in red blood cells. Am J Physiol 246: C195–203.
[4]
Yang B, Bankir L (2005) Urea and urine concentrating ability: new insights from studies in mice. Am J Physiol Renal Physiol 288: F881–896.
[5]
Bankir L, Chen K, Yang B (2004) Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement of urinary concentrating ability. Am J Physiol Renal Physiol 286: F144–151.
[6]
Sands JM, Gargus JJ, Frohlich O, Gunn RB, Kokko JP (1992) Urinary concentrating ability in patients with Jk(a-b-) blood type who lack carrier-mediated urea transport. J Am Soc Nephrol 2: 1689–1696.
[7]
Sidoux-Walter F, Lucien N, Olives B, Gobin R, Rousselet G, et al. (1999) At physiological expression levels the Kidd blood group/urea transporter protein is not a water channel. J Biol Chem 274: 30228–30235.
[8]
Yang B, Verkman AS (2002) Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes. J Biol Chem 277: 36782–36786.
[9]
Preston GM, Carroll TP, Guggino WB, Agre P (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385–387.
[10]
Murata K, Mitsuoka K, Hirai T, Walz T, Agre P, et al. (2000) Structural determinants of water permeation through aquaporin-1. Nature 407: 599–605.
[11]
Brown D, Verbavatz JM, Valenti G, Lui B, Sabolic I (1993) Localization of the CHIP28 water channel in reabsorptive segments of the rat male reproductive tract. Eur J Cell Biol 61: 264–273.
[12]
Heymann JB, Engel A (1999) Aquaporins: Phylogeny, Structure, and Physiology of Water Channels. News Physiol Sci 14: 187–193.
[13]
Wistow GJ, Pisano MM, Chepelinsky AB (1991) Tandem sequence repeats in transmembrane channel proteins. Trends Biochem Sci 16: 170–171.
[14]
Verbavatz JM, Brown D, Sabolic I, Valenti G, Ausiello DA, et al. (1993) Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study. J Cell Biol 123: 605–618.
[15]
Ko YJ, Huh J, Jo WH (2008) Ion exclusion mechanism in aquaporin at an atomistic level. Proteins 70: 1442–1450.
[16]
de Groot BL, Frigato T, Helms V, Grubmuller H (2003) The mechanism of proton exclusion in the aquaporin-1 water channel. J Mol Biol 333: 279–293.
[17]
de Groot BL, Grubmuller H (2005) The dynamics and energetics of water permeation and proton exclusion in aquaporins. Curr Opin Struct Biol 15: 176–183.
[18]
de Groot BL, Grubmuller H (2001) Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF. Science 294: 2353–2357.
[19]
Ilan B, Tajkhorshid E, Schulten K, Voth GA (2004) The mechanism of proton exclusion in aquaporin channels. Proteins 55: 223–228.
[20]
Fu D, Lu M (2007) The structural basis of water permeation and proton exclusion in aquaporins. Mol Membr Biol 24: 366–374.
[21]
Burykin A, Warshel A (2003) What really prevents proton transport through aquaporin? Charge self-energy versus proton wire proposals. Biophys J 85: 3696–3706.
[22]
Chakrabarti N, Tajkhorshid E, Roux B, Pomes R (2004) Molecular basis of proton blockage in aquaporins. Structure 12: 65–74.
[23]
Frohlich O, Macey RI, Edwards-Moulds J, Gargus JJ, Gunn RB (1991) Urea transport deficiency in Jk(a-b-) erythrocytes. Am J Physiol 260: C778–783.
[24]
Levin EJ, Cao Y, Enkavi G, Quick M, Pan Y, et al. (2012) Structure and permeation mechanism of a mammalian urea transporter. Proc Natl Acad Sci U S A 109: 11194–11199.
[25]
Levin EJ, Quick M, Zhou M (2009) Crystal structure of a bacterial homologue of the kidney urea transporter. Nature 462: 757–761.
[26]
Preston GM, Smith BL, Zeidel ML, Moulds JJ, Agre P (1994) Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels. Science 265: 1585–1587.
[27]
Saison C, Peyrard T, Landre C, Ballif BA, Schlosser KA, et al. (2012) A new AQP1 null allele identified in a Gypsy woman who developed an anti-CO3 during her first pregnancy. Vox Sang 103: 137–144.
[28]
Genetet S, Ripoche P, Picot J, Bigot S, Delaunay J, et al. (2012) Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells. Am J Physiol Cell Physiol 302: C419–428.
[29]
van Heeswijk MP, van Os CH (1986) Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine. J Membr Biol 92: 183–193.
[30]
Ye RG, Verkman AS (1989) Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes. Biochemistry 28: 824–829.
[31]
Mouro-Chanteloup I, Cochet S, Chami M, Genetet S, Zidi-Yahiaoui N, et al. (2010) Functional reconstitution into liposomes of purified human RhCG ammonia channel. PloS one 5: e8921.
[32]
Ripoche P, Bertrand O, Gane P, Birkenmeier C, Colin Y, et al. (2004) Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells. Proc Natl Acad Sci U S A 101: 17222–17227.
[33]
Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. Journal of molecular biology 234: 779–815.
[34]
Krivov GG, Shapovalov MV, Dunbrack RL (2009) Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77: 778–795.
[35]
Ray A, Lindahl E, Wallner B (2010) Model quality assessment for membrane proteins. Bioinformatics 26: 3067–3074.
[36]
Jorgensen WL, Chandrasekhar J, Madura JD (1983) Comparison of simple potential functions for simulating liquid water. The Journal of chemical physics 79.
[37]
Berendsen H, Postma J (1984) Molecular dynamics with coupling to an external bath. The Journal of chemical physics 81.
[38]
Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, et al. (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics (Oxford, England) 29: 845–854.
[39]
Cheatham TE 3rd, Cieplak P, Kollman PA (1999) A modified version of the Cornell et al. force field with improved sugar pucker phases and helical repeat. J Biomol Struct Dyn 16: 845–862.
[40]
Berger O, Edholm O, J？hnig F (1997) Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophysical journal 72: 2002–2013.
[41]
Tieleman DP, Maccallum JL, Ash WL, Kandt C, Xu Z, et al. (2006) Membrane protein simulations with a united-atom lipid and all-atom protein model: lipid-protein interactions, side chain transfer free energies and model proteins. J Phys Condens Matter 18: S1221–1234.
[42]
Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity rescaling. The Journal of chemical physics 126: 014101–014107.
[43]
Parrinello M, Rahman A (1981) Polymorphic transitions in single crystals: A new molecular dynamics method. Journal of Applied physics 52.
[44]
Hess B (2008) P-LINCS: A parallel linear constraint solver for molecular simulation. Journal of Chemical Theory and Computation 4: 116–122.
[45]
Cheatham TE III, Miller JL, Fox T, Darden TA, Kollman PA (1995) Molecular Dynamics Simulations on Solvated Biomolecular Systems: The Particle Mesh Ewald Method Leads to Stable Trajectories of DNA, RNA, and Proteins. Journal of the American Chemical Society 117: 4193–4194.
[46]
Beckstein O, Sansom MS (2003) Liquid-vapor oscillations of water in hydrophobic nanopores. Proc Natl Acad Sci U S A 100: 7063–7068.
[47]
Zhu F, Tajkhorshid E, Schulten K (2004) Theory and simulation of water permeation in aquaporin-1. Biophys J 86: 50–57.
[48]
Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 98: 10037–10041.
[49]
Olives B, Mattei MG, Huet M, Neau P, Martial S, et al. (1995) Kidd blood group and urea transport function of human erythrocytes are carried by the same protein. J Biol Chem 270: 15607–15610.
[50]
Mannuzzu LM, Moronne MM, Macey RI (1993) Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial. J Membr Biol 133: 85–97.
[51]
Agre P, Preston GM, Smith BL, Jung JS, Raina S, et al. (1993) Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol 265: F463–476.
[52]
Macey RI, Farmer RE (1970) Inhibition of water and solute permeability in human red cells. Biochim Biophys Acta 211: 104–106.
[53]
Yang B, Verkman AS (1998) Urea transporter UT3 functions as an efficient water channel. Direct evidence for a common water/urea pathway. J Biol Chem 273: 9369–9372.
[54]
Li H, Chen H, Steinbronn C, Wu B, Beitz E, et al. (2011) Enhancement of proton conductance by mutations of the selectivity filter of aquaporin-1. J Mol Biol 407: 607–620.
[55]
Nakhoul NL, Hering-Smith KS, Abdulnour-Nakhoul SM, Hamm LL (2001) Transport of NH(3)/NH in oocytes expressing aquaporin-1. Am J Physiol Renal Physiol 281: F255–263.
[56]
Geyer RR, Musa-Aziz R, Enkavi G, Mahinthichaichan P, Tajkhorshid E, et al. (2013) Movement of NH3 through the Human Urea Transporter B (UT-B): A New Gas Channel. Am J Physiol Renal Physiol 304 (12) F1447–F1457.
[57]
Zhao D, Sonawane ND, Levin MH, Yang B (2007) Comparative transport efficiencies of urea analogues through urea transporter UT-B. Biochim Biophys Acta 1768: 1815–1821.
[58]
Jensen MO, Dror RO, Xu H, Borhani DW, Arkin IT, et al. (2008) Dynamic control of slow water transport by aquaporin 0: implications for hydration and junction stability in the eye lens. Proc Natl Acad Sci U S A 105: 14430–14435.
[59]
Portella G, de Groot BL (2009) Determinants of water permeability through nanoscopic hydrophilic channels. Biophysical journal 96: 925–938.
[60]
Qiu H, Ma S, Shen R, Guo W (2010) Dynamic and energetic mechanisms for the distinct permeation rate in AQP1 and AQP0. Biochim Biophys Acta 1798: 318–326.
[61]
Mamonov AB, Coalson RD, Zeidel ML, Mathai JC (2007) Water and deuterium oxide permeability through aquaporin 1: MD predictions and experimental verification. J Gen Physiol 130: 111–116.
[62]
Hub JS, Grubmuller H, de Groot BL (2009) Dynamics and energetics of permeation through aquaporins. What do we learn from molecular dynamics simulations? Handb Exp Pharmacol 57–76.
[63]
Maciver B, Smith CP, Hill WG, Zeidel ML (2008) Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes. Am J Physiol Renal Physiol 294: F956–964.
[64]
Godara G, Smith C, Bosse J, Zeidel M, Mathai J (2009) Functional characterization of Actinobacillus pleuropneumoniae urea transport protein, ApUT. Am J Physiol Regul Integr Comp Physiol 296: R1268–1273.
[65]
Echevarria M, Frindt G, Preston GM, Milovanovic S, Agre P, et al. (1993) Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney. J Gen Physiol 101: 827–841.
[66]
Denker BM, Smith BL, Kuhajda FP, Agre P (1988) Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J Biol Chem 263: 15634–15642.
