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• W2011679999 abstract "Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, Lp, was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. TheLp values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeabilityPGl, obtained from [14C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (ς). For AQP3, water and glycerol interacted by competing for titratable site(s): ςGl was 0.15 at neutral pH but doubled at pH 6.4. The ς values were smaller for polyols in which the —OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol−1. We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites. Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, Lp, was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. TheLp values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeabilityPGl, obtained from [14C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (ς). For AQP3, water and glycerol interacted by competing for titratable site(s): ςGl was 0.15 at neutral pH but doubled at pH 6.4. The ς values were smaller for polyols in which the —OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol−1. We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites. Aquaporins (AQPs) 1The abbreviations used are: AQPs, aquaporins; EG, 1,2-ethanediol (ethylene glycol); PD, 1,2-pentanediol; Gl, 1,2,3-propanetriol (glycerol); B12, 1,2-butandiol; B13, 1,3-butandiol; B14, 1,4-butandiol; B23, 2,3-butandiol; P12, 1,2-pentandiol; P14, 1,4-pentandiol; P15, 1,5-pentandiol; P24, 2,4-pentandiol; MES, 2-(N-morpholino)ethanesulfonic acid.1The abbreviations used are: AQPs, aquaporins; EG, 1,2-ethanediol (ethylene glycol); PD, 1,2-pentanediol; Gl, 1,2,3-propanetriol (glycerol); B12, 1,2-butandiol; B13, 1,3-butandiol; B14, 1,4-butandiol; B23, 2,3-butandiol; P12, 1,2-pentandiol; P14, 1,4-pentandiol; P15, 1,5-pentandiol; P24, 2,4-pentandiol; MES, 2-(N-morpholino)ethanesulfonic acid. are a class of membrane proteins that allows osmotic water transport probably via an aqueous pore (1Agre P. Bonhivers M. Borgnia M.J. J. Biol. Chem. 1998; 273: 14659-14662Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Some AQPs can transport other solutes as well, AQP3 for example, supports significant fluxes of glycerol (2Echevarrı́a M. Windhager E.E. Tate S.S. Frindt G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10997-11001Crossref PubMed Scopus (266) Google Scholar, 3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar, 4Ma T. Frigeri A. Hasegawa H. Verkman A.S. J. Biol. Chem. 1994; 269: 21845-21849Abstract Full Text PDF PubMed Google Scholar, 5Echevarrı́a M. Windhager E.E. Frindt G. J. Biol. Chem. 1996; 271: 25079-25082Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 7Sasaki S. Ishibashi K. Marumo F. Annu. Rev. Physiol. 1998; 60: 199-220Crossref PubMed Scopus (66) Google Scholar). It has long been known that glycerol transport across the plasma membrane of the red blood cell is mediated by a pore and that the transport mechanism is inhibited at low pH (8Stein W.D. Biochim. Biophys. Acta. 1962; 59: 47-65Crossref PubMed Scopus (21) Google Scholar, 9Carlsen A. Wieth J.O. Acta Physiol. Scand. 1976; 97: 501-513Crossref PubMed Scopus (51) Google Scholar). Recently it was established that AQP3 is involved in glycerol transport in the red blood cell (10Roudier N. Verbavatz J.-M. Maurel C. Riproche P. Tacnet F. J. Biol. Chem. 1998; 273: 8407-8412Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). This raises the questions of whether glycerol transport in AQP3 is gated by H+ and whether water transport through AQP3 and other AQPs is also sensitive to H+.We have previously applied a fast and high resolution optical method to determine the transport properties of AQPs expressed inXenopus oocytes (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Here we combine this method with tracer measurements in order to study the effects of H+, temperature, and solute structure on transport of water, glycerol, and other straight chain polyols. The study is performed predominantly in AQP3, but also in AQP0, AQP1, AQP2, AQP4, and AQP5.At present, transport models are restricted to the use of macrophysical concepts such as pore diameter and pore length. This is mainly due to the lack of knowledge about the structure of the putative pore and the nature of the chemical interactions with the permeating molecules (11Finkelstein A. Water Movement through Lipid Bilyers, Pores and Plasma Membranes. Wiley-Interscience, New York1987Google Scholar,12Hill A.E. J. Membr. Biol. 1994; 137: 197-203Crossref PubMed Scopus (30) Google Scholar). Our data for AQP3 suggest a model where the permeation of water and polyols are determined by the formation of hydrogen bonds between the pore and the permeating molecule. From a physiological point of view, it is interesting that both the glycerol and the water transport through the AQP3 exhibited a strong, immediate, and reversible pH dependence. Such short term and direct gating of transport is a novel feature of aquaporins.MATERIALS AND METHODSDetails of the preparation of mRNA, the preparation and injection of Xenopus oocytes, and the set-up forLp and ς measurements have been described in detail previously (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 13Zeuthen T. Meinild A.-K. Klaerke D.A. Loo D.D.F. Wright E.M. Belhage B. Litman T. Biol. Cell. 1997; 89: 307-312PubMed Google Scholar, 14Meinild A.-K. Klaerke D.A. Loo D.D.F. Wright E.M. Zeuthen T. J. Physiol. (Lond.). 1998; 508.1: 15-21Crossref Scopus (162) Google Scholar). For measurements of the water permeability (Lp) and reflection coefficients (ς) oocytes were placed in a chamber (30 μl) in which solution changes could be accomplished within 5 s (90% complete). The oocytes were stabilized by the insertion of two microelectrodes, which also recorded the membrane potential. The presence of the microelectrodes did not affect the measurements (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Meinild A.-K. Klaerke D.A. Loo D.D.F. Wright E.M. Zeuthen T. J. Physiol. (Lond.). 1998; 508.1: 15-21Crossref Scopus (162) Google Scholar). Oocyte volumes were monitored on-line with an accuracy of 0.03% equal to about 0.4 nl, via an inverted microscope connected to a charge coupled device camera.Lp and ς were obtained from the initial rate of volume decrease induced by the addition of 20 mosmol of test solute to the control bathing solution. The ς of a given polyol was obtained from the ratio between the volume changes induced by the polyol and mannitol. The control bathing solution contained in mm: 90 NaCl, 20 mosmol of mannitol, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES or MES, osmolarity 214 mosmol; pH was adjusted according to the type of experiment. Test solutions were obtained by adding 20 mm mannitol or one of the following straight chain polyols (mole weights and boiling points (°C), respectively, in parentheses): ethylene glycol, EG (62, 197); 1,2-propanediol, PD (76, 187); glycerol, Gl (92, 193); 1,2-butandiol, B12 (90, 192); 1,3-butandiol, B13 (90, 204); 1,4-butandiol, B14 (90, 230); 2,3-butandiol, B23 (90, 184); 1,2-pentandiol, P12 (104, 206); 1,4-pentandiol, P14 (104, not detected); 1,5-pentandiol, P15 (104, 242), and 2,4-pentandiol, P24 (104, 201); the structures are indicated in Fig. 1B. The isotonic test solution was obtained by replacing 20 mosmol of mannitol in the control solution by 20 mosmol of glycerol.The glycerol permeability, PGl, was derived from [14C]glycerol uptakes. The oocytes were equilibrated for at least 2.5 min in the given test solution before being transferred for 0.5–10 min to 3 ml of the stirred test solution to which 4 μCi ml−1 [14C]glycerol (Amersham Pharmacia Biotech, code CFB 174, ethanol-free) had been added. To terminate uptake, oocytes were washed twice in ice-cold medium and transferred to scintillation vials, incubated for approximately 1 h at room temperature with 1 ml of 20% SDS before addition of 15 ml of scintillation fluid (Packard Opti-Fluor), and counting in a scintillation counter (Packard Tri-Carb).The Lp and PGl were analyzed as functions of external pH by fitting to a Boltzmann function: exp[(pH − pK)/ΔH]/(1 + exp[(pH − pK)/ΔH]). ΔH is a measure of the steepness of the curve around pK and is related to the Hill coefficient n = d(logLp)/d(pH) via n = 1/(2.3 ΔH).A phenomenological analysis of the volume and solute fluxes and the coupling between them was obtained from irreversible thermodynamics (15Katchalsky A. Curran P.F. Nonequilibrium Thermodynamics in Biophysics. Harvard University Press, Cambridge, MA1965Crossref Google Scholar), with glycerol as an example,JV=−A*R*T*Lp*(ΔCi+ςGl*ΔCGl)Equation 1 JGl=A*R*T*PGl*ΔCGl+JV*CGl′*(1−ςGl)Equation 2 where JV is the volume flow, determined from the initial rate of change in oocyte volume. JGlis the flux of glycerol, determined from tracer uptake. A is the true oocyte surface area: with an average diameter of 1.35-mm, oocytes have an apparent surface area of 5.9 mm2. Folding of the membrane increases this area by a factor of nine (16Zampighi G.A. Kreman M. Boorer K.J. Loo D.D.F. Bezanilla F. Chandy G. Hall J.E. Wright E.M. J. Membr. Biol. 1995; 148: 65-78Crossref PubMed Scopus (207) Google Scholar) to give the true surface area A = 0.53 cm2.R is the gas constant and T the absolute temperature. ΔCi is the transmembrane concentration difference of impermeable solutes such as mannitol; ΔCGl is the difference in glycerol concentration. CGl′ is the average concentration of glycerol in the aqueous pore. The coupling between the solute and volume fluxes in the aquaporin can be characterized by ς (Equations 10–56 in Ref. 15Katchalsky A. Curran P.F. Nonequilibrium Thermodynamics in Biophysics. Harvard University Press, Cambridge, MA1965Crossref Google Scholar),ςGl=1−R*T*PGl*(Δx/ϕw)*fswEquation 3 where Δx/ϕw is a constant that gives the ratio of the membrane thickness to the volume fraction of water,fsw is a formal frictional factor that gives the coupling between the solute (glycerol) and water in the pore. Changes in ς, which arise from partial molar volume effects, are ignored because they were too small to affect the measured values. It follows that a ς significantly smaller than 1 is evidence for interaction between solute and water and that 1 − ςGl is proportional to the glycerol permeability, PGl, times the friction, fsw. We will refer to this term as the interaction and relate Arrhenius activation energies (Ea) to this.The parameters presented have been corrected for the fluxes taking place via the membrane of the native oocytes. All numbers are given as means ± S.E., unless otherwise stated the number in parentheses is the number of experiments in at least four oocytes.DISCUSSIONAQP3 has been found to act as a channel for both water and glycerol transport. The fluxes are linear functions of their respective chemical driving forces (3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar, 5Echevarrı́a M. Windhager E.E. Frindt G. J. Biol. Chem. 1996; 271: 25079-25082Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 17Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), and the activation energies are low (3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar, 6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Furthermore, the two fluxes interact in the protein (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Our data show that the transports are gated by H+. In principle, three simultaneously active groups of titratable sites could be responsible for this behavior: one group in which titration led to closure of the channel(s), another that controlled theLp, and finally one that controlled thePGl. We will discuss the simplest possibility: that these groups are, at least partially, identical.Mechanisms and Coupling of Water and Glycerol FluxesThe behavior of AQP3 cannot be interpreted in terms of a physical pore. AQP3 remained open to glycerol transport in the pH range 5.8–6.2 while being closed for the smaller water molecule. The data can be interpreted by means of an Eyring energy barrier model (18Glasstone S. Laidler K.J. Eyring H. The Theory of Rate Processes. McGraw-Hill Book Co., Inc, New York1941Google Scholar). On this model, the molecule permeates by a series of jumps, the energy barriers of which are determined by the chemical bonds between the molecule and specific sites in the pathway. For AQP3, the Ea forLp was low, around 5 kcal mol−1, which suggests that the water molecule at neutral pH crosses this aquaporin by forming a succession of single hydrogen bonds. TheLp exhibited an immediate and reversible dependence on external pH (Fig. 2, A and B), under steady state conditions the Lp depended in a sigmoidal manner on external pH with a pK of 6.4 and a Hill coefficient of about 3. This suggests that at least three cooperating titratable sites determine the Lp. In the simplest, but not the only, model these sites are located in the aqueous pathway and determine the energy barriers for water permeation. Titration of the sites would abolish their hydrogen bonding capacity and render them effectively hydrophobic. In analogy to the Lp, thePGl had low activation energy and a marked dependence on external pH. But the pK was lower, 6.1, and the Hill coefficient larger, about 6 (Fig. 3B). This would suggest that glycerol also permeates by forming successive hydrogen bonds, the Hill coefficient indicates at least six.The difference between pK values and Hill coefficients raises the question whether it is the same titratable groups that determine the Lp and PGl. We suggest it is and that the difference arises from two effects. First, glycerol with its three —OH groups might have to make and break more hydrogen bonds than water in order to cross the aquaporin; this would lead to a higher Hill coefficient. Second, the pK for glycerol transport could be shifted due to a competitive interaction between H+ and glycerol at the sites. Such competition has been described in intact human red blood cells (9Carlsen A. Wieth J.O. Acta Physiol. Scand. 1976; 97: 501-513Crossref PubMed Scopus (51) Google Scholar) where glycerol transport is mediated by AQP3 (10Roudier N. Verbavatz J.-M. Maurel C. Riproche P. Tacnet F. J. Biol. Chem. 1998; 273: 8407-8412Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In these cells pK forPGl was about 6.0 at external glycerol concentrations of 1 mm and 5.5 at external glycerol concentrations of 2 m. In addition, the Hill coefficients were estimated to be larger than 2 (9Carlsen A. Wieth J.O. Acta Physiol. Scand. 1976; 97: 501-513Crossref PubMed Scopus (51) Google Scholar). These values are in agreement with those of the present study where glycerol concentrations of 20 mm were employed. It appears that glycerol, when close to the titratable site, to a certain extent displaces water molecules, an effect that would be enhanced by the confinement of the pore. The resulting lower molar fraction of water near the site would result in a lower local H+ concentration and consequently in a decrease of the effective pK. The hypothesis of a pathway shared by water and glycerol in AQP3 is supported by the finding that ςGl doubled when external pH was lowered to 6.4. This shows that the pathways for water and glycerol has at least one titratable site in common with a pK around 6.4. Titration reduces the availability of this site, and the interaction between the fluxes is reduced. At sufficiently acid pH values both water and glycerol would be unable to cross the channel. The fact that ς for formamide also doubled at pH 6.4 supports the notion that it is the —OH groups of the solute rather than its backbone that is responsible for interaction.Our model suggest a mechanism of how another member of the major integral protein family, the glycerol facilitator GlpF, can act as a glycerol channel without letting through water (19Maurel C. Reizer J. Schroeder J.I. Chrispeels M.J. Saier Jr., M.H. J. Biol. Chem. 1994; 269: 11869-11872Abstract Full Text PDF PubMed Google Scholar). If the pore of GlpF had a titratable site that was protonated at normal pH, it would be hydrophobic and in effect prevent the passage of water. If the site allowed competitive interaction between glycerol and H+ as described above, the glycerol molecule would be able to remove the H+ and use the site for transport.The model is not directly applicable to AQP0, AQP1, AQP2, AQP4, and AQP5, since they did not exhibit any pH sensitivity. One possibility is that the sites responsible for Lp in these aquaporins are not accessible to H+. AQP1 has a small but significant permeability to glycerol (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Abrami L. Tacnet F. Riproche P. Pflügers Arch. 1995; 430: 447-458Crossref PubMed Scopus (48) Google Scholar, 21Abrami L. Berthonaud V. Deen P.M.T. Rousselte G. Tacnet F. Riproche P. Pflügers Arch. 1996; 431: 408-414Crossref PubMed Scopus (35) Google Scholar), and the ς values for the smaller polyols, EG and PD, were of the same size as that of glycerol. This shows that although these polyols interacts with water in AQP1, some structural incompatibility, not found in AQP3, prevents them from permeating at any larger rate.The Role of Polyol CompositionIn general the ς values for AQP3 increased with the number of —OH groups and number of carbons of the test solute (Fig. 1B). The importance of —OH groups available for hydrogen bonding was particularly clear when ς values of the butanols B12, B13, B14, and B23 were compared. For these polyols the location and intramolecular interactions of the two —OH groups had significant specific effects on ς values. The ς values were larger if the two —OH groups were located next to each other and engaged in intramolecular bonding (ςB23 > ςB12 > ςB13 ≅ ςB14). The extent of intramolecular bonding was mirrored by the boiling points, which were lower for B12 and B23 than for B13 and B14. The —OH groups in B12 and B23 were therefore not available for interaction with the sites in the aquaporin to the same degree as the —OH groups of B13 and B14. This would result in smaller fluxes (smaller P) and/or smaller frictions with the water (smaller fsw) and therefore in larger ς values for B23 and B12 (Equation 3). The effects of the locations of the —OH groups on ς were absent for the pentanols. Most likely the longer carbon chain mitigate the strength of intramolecular bonding between —OH groups as witnessed by the small variations between the boiling points among this group.The picture that emerges is one where the test molecules, viewed as cylinders of different lengths and roughly similar diameters, cross the pore of AQP3 with their axis parallel to the pore. During permeation the —OH groups of the solute form a succession of single hydrogen bonds with the aquaporin as indicated by the low activation energies of around 5 kcal mol−1 observed forJGl, 1 − ςGl, 1 − ςB12, and 1 − ςB23.Comparison with Other StudiesThe numerical values for the transport parameters derived here and in an earlier paper (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) are compatible with the majority of published data of others (3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar, 5Echevarrı́a M. Windhager E.E. Frindt G. J. Biol. Chem. 1996; 271: 25079-25082Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Only one report gives a high Ea forPGl (5Echevarrı́a M. Windhager E.E. Frindt G. J. Biol. Chem. 1996; 271: 25079-25082Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar); we have no explanation for this discrepancy. The glycerol transport in AQP3 has been reported to be independent of external pH in the range 6–7 (22Ma T. Hasegawa H. Skach W.R. Frigeri A. Verkman A.S. Am. J. Physiol. 1994; 266: C189-C197Crossref PubMed Google Scholar), while we foundPGl to be significantly smaller at pH 6.0 (Fig. 3B). The same investigators (22Ma T. Hasegawa H. Skach W.R. Frigeri A. Verkman A.S. Am. J. Physiol. 1994; 266: C189-C197Crossref PubMed Google Scholar) also reported no water permeability of the AQP3, which is in contrast to all other reports (3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar,5Echevarrı́a M. Windhager E.E. Frindt G. J. Biol. Chem. 1996; 271: 25079-25082Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 17Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) and the present study.The ςGl determined by us in a previous study 0.24 (6Meinild A.-K. Klaerke D.A. Zeuthen T. J. Biol. Chem. 1998; 273: 32446-32451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) was higher than the one determined here, 0.15. The oocytes in the present study had more negative membrane potentials, on average −43 mV compared with the −25 mV in the previous study. As ςGlwas found to increase with acidity, we suggest that the oocytes used previously, being more stressed, might have had a more acid intracellular pH.Relation between Primary Structure and Permeation in AQP3The ability of AQP3 to transport both water and larger solutes is shared by AQP7 (24Ishibashi K. Kuwahara Y. Gu Y. Kageyama Y. Tohsaka A. Suzuki F. Marumo F. Sasaki S. J. Biol. Chem. 1997; 272: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 25Kuriyama H. Kawamoto S. Ishida N. Ohno I. Mita S. Matsuzawa Y. Matsubara K. Okubo K. Biochem. Biophys. Res. Commun. 1997; 241: 53-58Crossref PubMed Scopus (162) Google Scholar) and AQP9 (17Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Functionally, this places these three aquaporins in between those members of the major integral protein family that transport predominantly water (i.e. AQP0, AQP1, AQP2, AQP4, AQP5) and those that are impermeable to water,i.e. the glycerol facilitator GlpF (19Maurel C. Reizer J. Schroeder J.I. Chrispeels M.J. Saier Jr., M.H. J. Biol. Chem. 1994; 269: 11869-11872Abstract Full Text PDF PubMed Google Scholar). In view of the unique dependence of AQP3 on pH, titratable residues common for AQP3 and the water transporting aquaporins may not be relevant to explain the transport properties, while homology with the glycerol transporting aquaporins may be more important. If single residues are focused upon, an obvious guess for the titratable sites would be histidines, which qua their imidazole ring have pK values of 6.0–7.0 when incorporated into proteins. Other candidates with hydrophilic side groups are aspartate and glutamate residues, which may have pK values as high as 7 in proteins. All three amino acid residues are known to participate in hydrogen bonding (23Creighton T.E. Proteins. W. H. Freeman and Company, New York1993: 15Google Scholar).Interestingly, specific structural changes in aquaporins have been shown recently to cause shifts from water to glycerol permeation (26Lagrée V. Froger A. Deschamps S. Pellerin I. Delamarche C. Bonnec G. Gouranton J. Thomas D. Hubert J.-F. J. Biol. Chem. 1998; 273: 33949-33953Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,27Lagrée V. Froger A. Deschamps S. Hubert J.-F. Delmarche C. Bonnec G. Thomas D. Gouranton J. Pellerin I. J. Biol. Chem. 1999; 274: 6817-6819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). It should be investigated whether this phenomena and the one described by us have a common basis. The rapid and reversible effects of H+ observed by us, however, do not per seimplicate structural or major conformational changes.Physiological RelevanceAQP3 has been localized in several mammalian tissues: eye, kidney, stomach, spleen, intestine, and erythrocytes (3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar, 28Ecelbarger C.A. Terris J. Frindt G. Echevarrı́a M. Marpels D. Nielsen S. Knepper M.A. Am. J. Physiol. 1995; 269: F663-F672PubMed Google Scholar, 29Frigeri A. Gropper M.A. Turck C.W. Verkman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4328-4331Crossref PubMed Scopus (374) Google Scholar, 30Hamann S. Zeuthen T. La Cour M. Nagelhus E.A. Ottersen O.P. Agre P. Nielsen S. Am. J. Physiol. 1998; 274: C1332-C1345Crossref PubMed Google Scholar). For a recent review, see Ref. 7Sasaki S. Ishibashi K. Marumo F. Annu. Rev. Physiol. 1998; 60: 199-220Crossref PubMed Scopus (66) Google Scholar. The reduction of the Lp of AQP3 by H+suggests that these tissues under anaerobic conditions protect themselves against excessive cellular swelling by a reduction of the passive water permeability. The lower pK forPGl than for Lp shows that the cells strive to retain their capacity for glycerol uptake under acidosis. Aquaporins (AQPs) 1The abbreviations used are: AQPs, aquaporins; EG, 1,2-ethanediol (ethylene glycol); PD, 1,2-pentanediol; Gl, 1,2,3-propanetriol (glycerol); B12, 1,2-butandiol; B13, 1,3-butandiol; B14, 1,4-butandiol; B23, 2,3-butandiol; P12, 1,2-pentandiol; P14, 1,4-pentandiol; P15, 1,5-pentandiol; P24, 2,4-pentandiol; MES, 2-(N-morpholino)ethanesulfonic acid.1The abbreviations used are: AQPs, aquaporins; EG, 1,2-ethanediol (ethylene glycol); PD, 1,2-pentanediol; Gl, 1,2,3-propanetriol (glycerol); B12, 1,2-butandiol; B13, 1,3-butandiol; B14, 1,4-butandiol; B23, 2,3-butandiol; P12, 1,2-pentandiol; P14, 1,4-pentandiol; P15, 1,5-pentandiol; P24, 2,4-pentandiol; MES, 2-(N-morpholino)ethanesulfonic acid. are a class of membrane proteins that allows osmotic water transport probably via an aqueous pore (1Agre P. Bonhivers M. Borgnia M.J. J. Biol. Chem. 1998; 273: 14659-14662Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Some AQPs can transport other solutes as well, AQP3 for example, supports significant fluxes of glycerol (2Echevarrı́a M. Windhager E.E. Tate S.S. Frindt G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10997-11001Crossref PubMed Scopus (266) Google Scholar, 3Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi M. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref P" @default.
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