pH and Calcium Regulate the Water Permeability of Aquaporin 0

Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca2+ on water permeability ofXenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 ± 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 ± 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca2+ increased water permeability 4.1 ± 0.4-fold. The effects of Ca2+ and pH each required the presence of histidine 40, indicating a critical role of this amino acid in facilitating the modulation of water permeability. Clamping intracellular Ca2+ at high or low values abolished sensitivity to external Ca2+, suggesting that Ca2+ acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca2+ may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2–6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions. Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca2+ on water permeability ofXenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 ± 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 ± 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca2+ increased water permeability 4.1 ± 0.4-fold. The effects of Ca2+ and pH each required the presence of histidine 40, indicating a critical role of this amino acid in facilitating the modulation of water permeability. Clamping intracellular Ca2+ at high or low values abolished sensitivity to external Ca2+, suggesting that Ca2+ acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca2+ may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2–6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions. aquaporin 1,2-bis(2-aminophenoxy)ethane-N, N,N′,N′-tetraacetic acid diethylpyrocarbonate lysophosphatidic acid 2-(N-morpholino)ethanesulfonic acid N-(6aminohexyl)-5chloro-1-naphthalenesulfonamide The major intrinsic protein (MIP, now designated aquaporin 0 and abbreviated AQP0)1 of the optical lens was the first sequenced member of the aquaporins, an ancient family of proteins found in bacteria, plants, and animals (1.Gorin M.B. Yancey S.B. Cline J. Revel J.P. Horwitz J. Cell. 1984; 39: 49-59Abstract Full Text PDF PubMed Scopus (408) Google Scholar, 2.Pao G.M. Wu L.F. Johnson K.D. Hofte H. Chrispeels M.J. Sweet G. Sandal N.N. Saier Jr., M.H. Mol. Microbiol. 1991; 5: 33-37Crossref PubMed Scopus (150) Google Scholar, 3.Park J.H. Saier Jr., M.H. J. Membr. 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Work in several laboratories subsequently demonstrated that many members of the aquaporin family facilitate the diffusion of water and other nonelectrolytes (3.Park J.H. Saier Jr., M.H. J. Membr. Biol. 1996; 153: 171-180Crossref PubMed Scopus (262) Google Scholar, 8.Sasaki S. Ishibashi K. Marumo F. Annu. Rev. Physiol. 1998; 60: 199-220Crossref PubMed Scopus (66) Google Scholar, 9.Echevarria M. Kuang K.Y. Iserovich P. Li J. Preston G.M. Agre P. Fischbarg J. Am. J. Physiol. 1993; 265: C1349-C1355Crossref PubMed Google Scholar, 10.Ishida N. Hirai S.I. Mita S. Biochem. Biophys. Res. Commun. 1997; 238: 891-895Crossref PubMed Scopus (98) Google Scholar, 11.Kushmerick C. Rice S.J. Baldo G.J. Haspel H.C. Mathias R.T. Exp. Eye Res. 1995; 61: 351-362Crossref PubMed Scopus (76) Google Scholar, 12.Sabolic I. Valenti G. Verbavatz J.M. Van Hoek A.N. Verkman A.S. Ausiello D.A. Brown D. Am. J. Physiol. 1992; 263: C1225-C1233Crossref PubMed Google Scholar). Among the aquaporins, AQP0 forms a water channel with a relatively low water permeability (13.Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. Biol. 1997; 159: 29-39Crossref PubMed Scopus (158) Google Scholar, 14.Mulders S.M. Preston G.M. Deen P.M. Guggino W.B. van Os C.H. Agre P. J. Biol. Chem. 1995; 270: 9010-9016Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar); the water permeability per molecule is 40 times higher for AQP1 (13.Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. Biol. 1997; 159: 29-39Crossref PubMed Scopus (158) Google Scholar, 15.Yang B. Verkman A.S. J. Biol. Chem. 1997; 272: 16140-16146Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). The structural basis of this large difference in water permeability is unknown. AQP0 and AQP1 form tetrameric arrays in their native membranes and when reconstituted in lipid vesicles (6.Jung J.S. Bhat R.V. Preston G.M. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (627) Google Scholar, 7.Hasegawa H. Ma T. Skach W. Matthay M.A. Verkman A.S. J. Biol. Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar, 8.Sasaki S. Ishibashi K. Marumo F. Annu. Rev. Physiol. 1998; 60: 199-220Crossref PubMed Scopus (66) Google Scholar, 9.Echevarria M. Kuang K.Y. Iserovich P. Li J. Preston G.M. Agre P. Fischbarg J. Am. J. Physiol. 1993; 265: C1349-C1355Crossref PubMed Google Scholar, 10.Ishida N. Hirai S.I. Mita S. Biochem. Biophys. Res. Commun. 1997; 238: 891-895Crossref PubMed Scopus (98) Google Scholar, 11.Kushmerick C. Rice S.J. Baldo G.J. Haspel H.C. Mathias R.T. Exp. Eye Res. 1995; 61: 351-362Crossref PubMed Scopus (76) Google Scholar, 12.Sabolic I. Valenti G. Verbavatz J.M. Van Hoek A.N. Verkman A.S. Ausiello D.A. Brown D. Am. J. Physiol. 1992; 263: C1225-C1233Crossref PubMed Google Scholar, 13.Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. 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Van Boxtel H.A. Van Os C.H. Deen P.M. Pflugers. Arch. Eur. J. Physiol. 1998; 436: 599-607Crossref PubMed Scopus (10) Google Scholar). Although low in water permeability per molecule, AQP0 comprises more than 60% of the membrane protein in the normal vertebrate lens and therefore provides the major permeability pathway for water movement across the membranes of lens fiber cells. If it is defective or missing from an otherwise normal lens, a cataract results (20.Shiels A. Griffin C.S. Curr. Eye Res. 1993; 12: 913-921Crossref PubMed Scopus (31) Google Scholar, 21.Shiels A. Bassnett S. Nat. Genet. 1996; 12: 212-215Crossref PubMed Scopus (221) Google Scholar). In a chimeric mouse model, cataract can be prevented by the presence of 20% normal cells, which presumably supply the requisite AQP0 (22.Muggleton-Harris A.L. Hardy K. Higbee N. Development. 1987; 99: 473-480PubMed Google Scholar). The role of AQP0 in maintaining normal lens conditions is uncertain, but it likely facilitates the intrinsic circulation of fluid in the lens that maintains lens transparency and homeostasis in the absence of blood vessels (23.Mathias R.T. Rae J.L. Baldo G.J. Physiol. Rev. 1997; 77: 21-50Crossref PubMed Scopus (345) Google Scholar). pH and Ca2+ are likely candidates for effecting regulatory control of this circulation, because both of these ions seem to play important roles in the lens. The lens interior is more acidic (pH 6.5) than the surface (pH 7.02) (24.Mathias R.T. Riquelme G. Rae J.L. J. Gen. Physiol. 1991; 98: 1085-1103Crossref PubMed Scopus (72) Google Scholar, 25.Pasquale L.R. Mathias R.T. Austin L.R. Brink P.R. Ciunga M. Biophys. J. 1990; 58: 939-945Abstract Full Text PDF PubMed Scopus (9) Google Scholar), and disturbances in Ca2+ concentration are associated with cataract (26.Duncan G. Jacob T.J.C. Nugent J. Whelan J. Human Cataract Formation. Pitman Press, Bath, UK1984: 132-152Google Scholar, 27.Paterson C.A. Zeng J. Husseini Z. Borchman D. Delamere N.A. Garland D. Jimenez-Asensio J. Curr. Eye Res. 1997; 16: 333-338Crossref PubMed Scopus (68) Google Scholar, 28.Takehana M. Exp. Eye Res. 1990; 50: 671-676Crossref PubMed Scopus (29) Google Scholar). In this paper we show that both pH and Ca2+ can regulate the water permeability of AQP0 expressed in Xenopus oocytes but not the water permeability of AQP1 or AQP4. We localize the molecular site of pH modulation to a single extracellular histidine, His40, unique to AQP0. Modulation of water permeability by local ionic changes within the lens interior may play an important role in lens physiology. Some of the results reported here were previously presented in abstract form (29.Cahalan K. Hall J.E. J. Gen. Physiol. 1998; 112: 72Google Scholar, 30.Cahalan K.L. Hall J.E. Biophys. J. 1998; 76 (abstr.): 183Google Scholar). Female Xenopus laeviswere anesthetized, and stage V and VI oocytes removed and prepared as described previously (13.Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. Biol. 1997; 159: 29-39Crossref PubMed Scopus (158) Google Scholar). The day after isolation, oocytes were injected with either 10 ng of AQP0 or 5 ng of AQP1 cRNA (except as noted) and maintained in ND96 (96 mm NaCl, 2 mmKCl, 1 mm MgCl2, 1.8 mmCaCl2, 5 mm HEPES, pH 7.5) supplemented with 10 mg/ml penicillin, 10 mg/ml streptomycin, and 2.5 mm sodium pyruvate at 18 °C. The expression constructs for bovine AQP0 and human AQP1 were gifts from Peter Agre and Greg Preston (Johns Hopkins). The rat AQP4 gene was purchased from ATCC (number 87184) and placed in the same expression vector. RNA was transcribed in vitro using T3 RNA polymerase (mMESSAGE mMACHINE kit, Ambion). Histidine was substituted by alanine, aspartate, or lysine at position 40 in AQP0, using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, the mutants were obtained by performing a one step polymerase chain reaction with a set of two appropriate primers overlapping in the region of the mutation using PfuTurbo DNA polymerase. The mutations were confirmed by sequencing using fluorescent dye terminators (University of Chicago, DNA Sequencing Facility). After 2 days, oocyte swelling assays were performed at 15 °C by transfer from 100% ND96 to 30% ND96. Before the transfer to 30% ND96 at the experimental pH or Ca2+ concentration, oocytes were always equilibrated for 5 min in 100% ND96 at the same experimental pH or Ca2+ concentration. Water permeability in cm/s,P f, was calculated as described previously from optical measurements of the increase in cross-sectional area of the oocyte with time in response to a challenge with diluted ND96 (13.Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. Biol. 1997; 159: 29-39Crossref PubMed Scopus (158) Google Scholar). Because the water permeability of uninjected oocytes has a very high activation energy, about 25 kcal/mol, whereas the activation energy of the water permeability induced by the aquaporins is small, 4–8 kcal/mole (13.Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. Biol. 1997; 159: 29-39Crossref PubMed Scopus (158) Google Scholar, 14.Mulders S.M. Preston G.M. Deen P.M. Guggino W.B. van Os C.H. Agre P. J. Biol. Chem. 1995; 270: 9010-9016Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), a signal to noise advantage is obtained by performing these measurements at 15 °C rather than 20 °C. Unless otherwise noted, each data point is the average of experiments using nine oocytes from three different batches. Because AQP0 has a lower permeability than other aquaporins, we used relative permeability to facilitate comparison and to correct for background. We define relative water permeability as: relative Pf=Pexp−PctPst−PUIEquation 1 where P exp is the permeability measured under experimental P is the permeability measured under conditions of pH 1.8 mm Ca2+, and P is the permeability of uninjected oocytes. The of is calculated from the of the using the for the of P.R. and for the Scholar). uninjected oocytes an average water permeability of ± and change in water permeability under of the experimental conditions in Ca2+ concentration, calmodulin We a of these control data in 1 but we not show the uninjected control results In where we show data for oocytes injected with AQP1 and two different aquaporins water permeability not change under the experimental conditions. each experimental pH 100% and 30% ND96 were using for pH to pH and for pH 6.5 to pH Before the swelling in 30% ND96 at the experimental the oocytes were in 100% ND96 at the experimental pH for 5 were in a mm at pH for 5 the oocytes were for 5 min at pH to the the oocytes were for 5 min at pH and the swelling was performed under appropriate experimental conditions. were in a of pH 7.5) for min and in pH for 5 min performing the swelling each experimental Ca2+ concentration, 100% and 30% ND96 were as 1 mm 1.8 mm Ca2+ or 10 mm Before the swelling was performed in 30% ND96 at the experimental Ca2+ concentration, the oocytes were in 100% ND96 at the experimental Ca2+ concentration for 5 20 of mm were injected the oocyte by min the swelling assays were The concentration in the oocyte was about 2 were in for min performing the swelling in ND96 pH with or 1.8 mm were in 5 or N-(6aminohexyl)-5chloro-1-naphthalenesulfonamide for min in the performing the swelling at pH 1.8 mm Ca2+, with the concentration maintained at the We investigated the effects of external pH and on the water permeability induced in oocytes by the AQP0, for AQP1 and AQP4. external ionic conditions of pH and 1.8 the water permeability of AQP0 is lower than that for when pH is the water permeability of AQP0 whereas that of AQP1 and uninjected oocytes 1 The relative permeability, calculated to (1.Gorin M.B. Yancey S.B. Cline J. Revel J.P. Horwitz J. Cell. 1984; 39: 49-59Abstract Full Text PDF PubMed Scopus (408) Google Scholar), is 3.4 ± at pH 6.5 for AQP0. 1 a for the water of AQP0, AQP1, and uninjected oocytes as relative The for AQP0 has an a of than a water permeability at pH and a at pH is not a of pH but as the pH is suggesting the of at one site. the of found at pH may and reported pH of AQP0 by at pH and T. D.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). effects of pH on AQP0 water permeability were within the of time required to solutions. The expression of AQP0 has on the pH the pH modulation for two different expression of AQP0, by the of In either the pH to 6.5 increased the AQP0 water permeability by a of about the of water permeability the pH sensitivity, we the expression of AQP1 and AQP4 to of water permeability with with 10 ng of injected AQP0 1 that at expression effects of pH on AQP1 or AQP4 water permeability were We that the water permeability of AQP0 is increased by low pH and that this increase is not by either expression or of water permeability. The pH of AQP0 water permeability the of one or more histidine this we investigated the of on water permeability. can histidine, or amino acid but histidine when the reaction is at pH the reaction with histidine, but not with lysine or can be by J.E. and Scholar). pretreatment increased relative P to 4.2 ± 1 with In pretreatment abolished the pH sensitivity of the AQP0 water permeability 2 indicating that results that low pH and act at the same site or to increase AQP0 water permeability. The effects of on AQP0 water permeability were by with that the are pretreatment on the water permeability of oocytes 2 pretreatment increased the water permeability of AQP0, with on the water permeability of oocytes or uninjected control oocytes not these we that a histidine or an role in pH modulation of AQP0 water permeability. Because the increase in water permeability induced by low pH was to AQP0 and not for AQP1 and we the of these aquaporins to for histidine the membrane of AQP0, the as the membrane as by the AQP0 has a histidine in each extracellular and AQP1 has the and AQP4 has the and the and T. K. K. M. K. S. 1996; PubMed Scopus Google Scholar). the histidine. this we three different mutants of AQP0, by acid or lysine of these mutants was and in and each water permeability under conditions two times than that of under conditions of cRNA and and water permeability experiments show that an role in the pH sensitivity of the water permeability of of pH and on AQP0 mutants water permeability. three mutants and different and of the mutants water to that of AQP0 at pH water on the three histidine Unless otherwise each data point is the average of nine measurements different of three oocytes from each for of we investigated the effects of pretreatment on the three AQP0, of the mutants change in water permeability with We that histidine at position 40 is for the pH modulation of water permeability in AQP0. The in the and extracellular are either to by or have in the pH and Ca2+ are in of membrane and therefore we effects of extracellular and pH and in at pH increased relative AQP0 water permeability by a of 4.1 ± 5 but on the relative water permeability of oocytes 5 or uninjected control oocytes not to the effects of the increased water permeability induced by Ca2+ was to ionic conditions. of to Ca2+ ions a than Ca2+, suggesting a to that when pH was Ca2+ to 10 mm with ionic conditions. 5 that the AQP0 water permeability a range by the of cRNA injected not the relative permeability in low Ca2+ with conditions. results that external AQP0 water permeability by about the same as is the range of water permeability modulation that can be by that the on AQP0 water permeability of pH and Ca2+ is than pH or Ca2+ but the effects are not Lowering pH and Ca2+ increased the relative permeability to ± the of increase in experiments but than the of the relative to pH ± and ± results the of two different of for of pH and to the of Ca2+ on the histidine mutants pH that three histidine mutants to the increase in water permeability by under low Ca2+ conditions. that pH or Ca2+ the water permeability a low permeability under and a higher permeability The of low Ca2+ to the permeability of of the mutants that this the critical histidine 40 the effects of low Ca2+ are mediated or the we the internal Ca2+ at low values using and at high values using the internal Ca2+ in the oocyte and the internal Ca2+ PubMed Scopus Google Scholar). that injected concentration, 2 increased the water permeability induced by AQP0 under ionic conditions. the water permeability to change induced by external of not the water permeability of AQP1 or control uninjected oocytes not through a internal S. K. N. T. T. Biochem. Biophys. Res. 1993; PubMed Scopus Google Scholar, S. M. and J. K. J. Cell. Physiol. Scholar). in the the water permeability at control values for AQP0 and the in permeability induced by low external Ca2+ We that and the increased water permeability in AQP0 induced by external In two laboratories reported that calmodulin with AQP0 called major intrinsic S.J. C. Curr. Eye Res. 1991; PubMed Scopus Google Scholar, P. G. Exp. Eye Res. 1990; 50: PubMed Scopus Google Scholar), suggesting the that calmodulin the changes in water permeability in AQP0. We three calmodulin and P. F. G.A. J. Am. 1996; Google Scholar) and found that each increased the AQP0 water permeability by an average of ± and it to of the external inhibitors on the water of AQP1 or uninjected control oocytes not The that three calmodulin inhibitors have the same on water permeability that the increase in water permeability may be mediated by calmodulin. In this we that the external pH or Ca2+ AQP0 water permeability and that is for the protein to high and low permeability modulation in permeability may be important in the lens, increased water circulation times of increased results with a that AQP0 change in water permeability and that water permeability to at acid pH K.L. Hall J.E. Biophys. J. 1998; 76 (abstr.): 183Google Scholar). AQP0 and are the aquaporins that share the three His40, and in AQP0. results the that may facilitate in as We the conditions the high or low permeability of AQP0 in with the in experimental increased AQP0 water permeability by a of three to The effects of pH and Ca2+ were than suggesting of a as single channel permeability or the of channel the mutants not pH sensitivity but sensitivity, suggesting that the AQP0 water permeability two one and one that a His40, to the range of of AQP0 water Ca2+ and pH pH ± ± mutants ± mutants the permeability in P was that of AQP0 at pH 1.8 mm and low ± Ca2+ and low pH ± ± and low Ca2+ ± inhibitors ± the conditions that high or low water permeability of AQP0. The in show the relative permeability calculated to 1 or the mutants the permeability in P was that of AQP0 at pH 1.8 mm in a the conditions that high or low water permeability of AQP0. The in show the relative permeability calculated to 1 or the The and with data from the calmodulin external and internal Ca2+ concentration in the oocyte We this at because the internal concentration at high or low values the of external Ca2+, whereas the is not we that the Ca2+ concentration be The of this paper is that the water permeability of AQP0 is by ions in the physiological range found in the lens. data that AQP0, but not AQP1 or can from low to high permeability as pH or Ca2+ is Because AQP0 is the major membrane protein of the lens and at a very high is the to increase the water permeability of a fiber pH the water permeability of AQP0 in the interior of the lens. Because pH is a of it a signal to increase fluid to of increased or to by the intrinsic We for oocyte and and K. for