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Nickel is a common cause of pneumoconiosis. Here, we show that nickel inactivates aquaporin (AQP)-3, the water channel expressed apically in epithelial cells of human terminal airways. Human AQP3 was transiently transfected into human lung cells, and water permeability was measured in transfected and neighboring untransfected cells. Incubation with NiCl2 rapidly, dose-dependently, and reversibly decreased water permeability in AQP3-expressing cells. Acidification of the extracellular medium also caused rapid, dose-dependent, and reversible inhibition of AQP3. Sensitivity of AQP3 to nickel was lower at alkaline pH than at neutral and acidic pH. Cells transfected with human AQP4 and AQP5, which are also expressed in airway epithelia, were insensitive to nickel and extracellular acidification. Zinc and cadmium, other common causes of pneumoconiosis, had no effect on the water permeability of AQP3. Three extracellular residues, Trp128, Ser152, and His241, were responsible for the blocking effect of nickel on human AQP3. Ser152 was identified as a common site for nickel and pH sensitivity. His53, Tyr124, and His154 were also involved in regulation of AQP3 by extracellular pH. In addition, the aromatic side chain of His154 was shown to be important for the water permeability of AQP3. Our results imply that nickel and extracellular pH may modulate lung water clearance and that defective water clearance may be an early component of nickel-induced lung disease. Nickel is a common cause of pneumoconiosis. Here, we show that nickel inactivates aquaporin (AQP)-3, the water channel expressed apically in epithelial cells of human terminal airways. Human AQP3 was transiently transfected into human lung cells, and water permeability was measured in transfected and neighboring untransfected cells. Incubation with NiCl2 rapidly, dose-dependently, and reversibly decreased water permeability in AQP3-expressing cells. Acidification of the extracellular medium also caused rapid, dose-dependent, and reversible inhibition of AQP3. Sensitivity of AQP3 to nickel was lower at alkaline pH than at neutral and acidic pH. Cells transfected with human AQP4 and AQP5, which are also expressed in airway epithelia, were insensitive to nickel and extracellular acidification. Zinc and cadmium, other common causes of pneumoconiosis, had no effect on the water permeability of AQP3. Three extracellular residues, Trp128, Ser152, and His241, were responsible for the blocking effect of nickel on human AQP3. Ser152 was identified as a common site for nickel and pH sensitivity. His53, Tyr124, and His154 were also involved in regulation of AQP3 by extracellular pH. In addition, the aromatic side chain of His154 was shown to be important for the water permeability of AQP3. Our results imply that nickel and extracellular pH may modulate lung water clearance and that defective water clearance may be an early component of nickel-induced lung disease. Nonenzymatic regulation of ion channels by Ni2+ and other divalent cations or by pH is a well established phenomenon with many important physiological and pathophysiological implications. Less is known about nonenzymatic regulation of water channels, aquaporins (AQPs) 1The abbreviations used are: AQPs, aquaporins; GFP, green fluorescent protein; P f, water permeability; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid.1The abbreviations used are: AQPs, aquaporins; GFP, green fluorescent protein; P f, water permeability; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid. (1Agre P. King L.S. Yasui M. Guggino W.B. Ottersen O.P. Fujiyoshi Y. Engel A. Nielsen S. J. Physiol. (Lond.). 2002; 542: 3-16Crossref Scopus (915) Google Scholar). Mercury inhibits most mammalian water channels via binding to cysteine residues (2Preston G.M. Jung J.S. Guggino W.B. Agre P. J. Biol. Chem. 1993; 268: 17-20Abstract Full Text PDF PubMed Google Scholar, 3Zhang R. van Hoek A.N. Biwersi J. Verkman A.S. Biochemistry. 1993; 32: 2938-2941Crossref PubMed Scopus (126) Google Scholar, 4Kuwahara M. Gu Y. Ishibashi K. Marumo F. Sasaki S. Biochemistry. 1997; 36: 13973-13978Crossref PubMed Scopus (91) Google Scholar) and has been an important tool in studies of AQPs. Gold and silver were recently reported to inhibit a water channel from human erythrocytes, presumably AQP1, but a molecular basis for this inhibition has not been revealed (5Niemietz C.M. Tyerman S.D. FEBS Lett. 2002; 531: 443-447Crossref PubMed Scopus (245) Google Scholar). The question of whether Ni2+ and other divalent ions known to regulate the activity of ion channels modulate the activity of AQPs has, to our knowledge, not yet been addressed. Nickel is widely used in modern industry (reviewed in Ref. 6Kelleher P. Pacheco K. Newman L.S. Environ. Health. Perspect. 2000; 108: 685-696Crossref PubMed Scopus (151) Google Scholar). Inhalation is the primary route of occupational exposure to nickel and other heavy metals, and inhalation of nickel compounds is a common cause of pneumoconiosis (6Kelleher P. Pacheco K. Newman L.S. Environ. Health. Perspect. 2000; 108: 685-696Crossref PubMed Scopus (151) Google Scholar, 7Nemery B. Eur. Respir. J. 1990; 3: 202-219PubMed Google Scholar). AQP3, AQP4, and AQP5 are expressed in the airway epithelia (8Nielsen S. King L.S. Christensen B.M. Agre P. Am. J. Physiol. 1997; 273: C1549-C1561Crossref PubMed Google Scholar, 9Matsuzaki T. Suzuki T. Koyama H. Tanaka S. Takata K. J. Histochem. Cytochem. 1999; 47: 1275-1286Crossref PubMed Scopus (143) Google Scholar, 10Kreda S.M. Gynn M.C. Fenstermacher D.A. Boucher R.C. Gabriel S.E. Am. J. Respir. Cell Mol. Biol. 2001; 24: 224-234Crossref PubMed Scopus (218) Google Scholar). AQP3 is located at the apical membrane of human lung epithelium (10Kreda S.M. Gynn M.C. Fenstermacher D.A. Boucher R.C. Gabriel S.E. Am. J. Respir. Cell Mol. Biol. 2001; 24: 224-234Crossref PubMed Scopus (218) Google Scholar). Here, we have examined the effects of Ni2+ on the water permeability of human AQP3, AQP4, and AQP5 expressed in a human lung cell line. Since AQP3 has, when expressed in oocytes, been reported to be pH-sensitive (11Zeuthen T. Klaerke D.A. J. Biol. Chem. 1999; 274: 21631-21636Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), we also examined the effect of extracellular acidification. We show that Ni2+ and pH regulate the water permeability of human AQP3, but not of human AQP4 and AQP5. We also address the question of whether Ni2+ and pH may interact in the regulation of human AQP3. Identification of the molecular sites responsible for the Ni2+ and pH sensitivity of AQP3 is important for future development of therapeutic agents. Histidine, with a pK a of ∼6.5, is the most likely molecular target for regulation by pH and is also a preferential site for Ni2+ binding. By performing a series of mutations of extracellular histidines and amino acids considered to interact with histidine, we identified several molecular determinants of pH and Ni2+ sensitivity and at least one common determinant of Ni2+ and pH sensitivity. DNA Constructs—cDNA fragments encoding full-length AQP3 and the long form of AQP4 were obtained by amplification from the human lung QUICK-Clone cDNA library (Clontech). cDNA encoding human AQP5 was a generous gift from P. Agre (Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD). The cDNA fragments were used for creation of two types of cDNA constructs: constructs that expressed a water channel fused with green fluorescent protein (GFP) and constructs that expressed a water channel and GFP as separate proteins present in the same cell. For the first type of construct, cDNA fragments were subcloned in-frame into the pEGFP-N2 vector for AQPs tagged with GFP at the COOH terminus and in-frame into the pEGFP-C2 vector for AQPs tagged with GFP at the NH2 terminus. For the second type of construct, cDNA fragments were subcloned into the pIRES2-EGFP vector (Clontech). The point mutations in the extracellular loops of human AQP3 were generated by PCR-based mutagenesis using wild-type cDNA as a template. The presence of each point mutation and absence of other modifications were confirmed by sequence analysis of the whole insert. The transmembrane structure of human AQP3 was predicted using TMHMM Version 2.0 (12Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9084) Google Scholar). The protein sequences were aligned with ClustalW Version 1.81 (13Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar). Cell Culture—The human bronchial epithelial cell line BEAS-2b (subpassages 10–36; European Collection of Cell Cultures, Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK) was cultured on coverslips (Bioptechs, Butler, PA) coated with collagen type I and fibronectin (Sigma, Stockholm, Sweden) in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (1:1; Invitrogen, Paisley, Scotland, UK) containing 0.5 units/ml penicillin and 50 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal bovine serum and 2 mm l-glutamine. On the second day of culture, the cells were transiently transfected with cDNA constructs (see above) using CLONfectin (Clontech) according to the manufacturer's protocol. Experiments were performed on the fourth day of culture. of water permeability was measured using a that we recently in M. S. H. A. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). The one to the P in cells cell and to the P in cells that and not the coverslips with the transfected cells were in a Cell Butler, PA) on the of a and 2 with at and at the of P an the of proteins was with the on the of the the cells were with The the P f, the cells were with mm mm mm mm mm and mm pH The was to was decreased by the of to the P at a the pH of the and was to the In at pH and and were In with and in were with mm and mm was to The P was using the M. S. H. A. Am. J. Physiol. 2002; PubMed Scopus Google P The was for cell from the in the cell the of the cell was using performed using the on of the cells at and the same cells in For BEAS-2b cells, the of the cell was to be The was as the of the the For BEAS-2b cells, was to be is the cell was using on of the cells in For BEAS-2b cells, the was to be is the of water and is the which was in our The P in BEAS-2b cells and the P in transfected cells on the of CLONfectin used for the and the cell we the P in cells that were at a and transfected with the same CLONfectin of the of whether the effects of acidic pH or NiCl2 were to a of in the the transfected cells were in the same as used for P in the of with a of was The was to a pH or a containing mm and a of was The of GFP in the membrane to that in the was measured using of MD). at The of AQP3 was in the same cells in two at the same cell were for the or of the cells was in to pH or of were with the pH-sensitive fluorescent by with for at The cells were in the same used for P (see above) on a using a was at of and was with a were with a to a and using from the of each the pH was using containing mm mm or and to pH and and are as S.E. analysis was performed using of was considered and of Human AQP3 into AQP3 was transiently transfected into a bronchial epithelial cell line GFP was used to cells AQP3. In cells transfected with AQP3 tagged with GFP at the COOH terminus a GFP was in the the was or in the The membrane was the apical and of the cells In cells transfected with AQP3 and GFP as separate proteins the GFP was the and neutral and alkaline cells or had a P than neighboring cells a GFP we that the protein was a water Sensitivity of Human AQP3 to pH and of the extracellular decreased the P in cells untransfected cells pH the P in transfected cells was not from that in untransfected cells. The pH effect was in cells and in cells of water permeability by acidic pH was and reversible and was no in the of human in cells to extracellular pH. The of GFP in to that in the was and in pH when the water permeability was the pH was when the extracellular pH was from to and Nickel also decreased the P in cells human in a The effect was and reversible The P in untransfected cells was not by was no in the of in cells to The of GFP in to that in the was and in with mm when the water permeability was The effect of Ni2+ was neutral and acidic the water permeability was by mm pH and the P in transfected cells was decreased by but than that in untransfected cells. of the pH and on Human AQP4 and AQP5 AQP3, expressed in the epithelial cells of the lower of the human lung (10Kreda S.M. Gynn M.C. Fenstermacher D.A. Boucher R.C. Gabriel S.E. Am. J. Respir. Cell Mol. Biol. 2001; 24: 224-234Crossref PubMed Scopus (218) Google Scholar). BEAS-2b cells were transfected with human AQP4 or AQP5 using cDNA constructs encoding GFP and the water channels as separate or The water permeability was in cells AQP4 or AQP5 with untransfected cells. the extracellular pH to or cells to Ni2+ had no effect on the P a and and are well causes of pneumoconiosis (6Kelleher P. Pacheco K. Newman L.S. Environ. Health. Perspect. 2000; 108: 685-696Crossref PubMed Scopus (151) Google Scholar, 7Nemery B. Eur. Respir. J. 1990; 3: 202-219PubMed Google Scholar). Cells transfected with were with mm or The P in cells or in untransfected cells was not by of compounds of the and pH Sensitivity of Human the molecular determinants of Ni2+ and pH we performed a series of point mutations in the extracellular loops of AQP3 The extracellular loops were for the the regulation of AQP3 by Ni2+ and pH was and the in extracellular pH that the water permeability of AQP3 not to in pH. inhalation is the route for Ni2+ AQP3 is expressed apically in the human and extracellular loops be the first target for nickel binding. The amino acids were for histidine, which interact with Ni2+ S. M. Biol. PubMed Scopus Google Scholar) and which is the amino with a pK a in the for AQP3 pH and may interact with Biochemistry. PubMed Scopus Google Scholar, D.A. Biochemistry. 1993; 32: PubMed Scopus Google Scholar, A. T. H. Biochemistry. 2001; PubMed Scopus Google Scholar). The results from the mutation studies are in and the most results are in and and a and were to the of the cells. In two and AQP3 was In and the water permeability in cells AQP3 was decreased with that in cells wild-type of AQP3 for pH and in pH sensitivity was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected effect of Ni2+ was effect of Ni2+ was was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected effect of Ni2+ was was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected was not from the in untransfected was not from the in untransfected effect of Ni2+ not be measured the was not from the in untransfected The was not from the in untransfected The effect of Ni2+ not be measured the was not from the in untransfected The effect of Ni2+ was in a the pH sensitivity of human AQP3. are as the P in transfected cells and the untransfected cells from the same coverslips the P in cells or was lower than that in cells wild-type AQP3 The of pH sensitivity was to pH in cells the mutation human AQP3 The P in cells was to that in cells wild-type AQP3, but was at pH the mutation decreased the water permeability of AQP3 and the pH sensitivity to pH the water permeability in cells was to acidic pH. pH the P in transfected cells was than that in untransfected cells, in cells wild-type AQP3, the water P was Three residues, and Ser152 in the second extracellular and in the extracellular were identified as determinants of AQP3 Ni2+ sensitivity and The P in cells or was to that in cells wild-type AQP3. Ni2+ had no effect on the water permeability of of of AQP3 the pH of the water In cells or the of pH sensitivity was to alkaline In cells the of pH sensitivity was to acidic pH The water permeability of wild-type AQP3 was at pH this the P in cells was with that in the untransfected cells. The of membrane to a on to be Here, we show that the water permeability of human AQP3 expressed in human lung cells is by in extracellular pH and by Ser152 in the second extracellular of AQP3 was identified as a common determinant of Ni2+ and pH sensitivity. Our have several important for the of lung and as well as for the of other in which AQP3 is as the is well that Ni2+ or the activity of ion channels S.E. Full Text PDF PubMed Scopus Google Scholar, J. Biol. 1996; PubMed Scopus Google Scholar, J. Biol. 2001; PubMed Scopus Google Scholar, M. S. Y. Y. J. Physiol. 2002; PubMed Scopus Google Scholar, S. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), but this to the of our knowledge, the first of Ni2+ regulation of the activity of a water The effect of Ni2+ was and on at least extracellular In studies of the by which Ni2+ the activity of channels and the epithelial extracellular histidines were to be the molecular determinants of Ni2+ sensitivity S.E. Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). in the side chain of is the target for Ni2+ binding S. M. Biol. PubMed Scopus Google Scholar). Human AQP3 has one in the first extracellular two in the and one in the In this we that one of extracellular residues, His241, is for the Ni2+ sensitivity of human AQP3. The side chain of has a structure that is to that of the of we that may also be a for Ni2+ binding. we that of with the Ni2+ sensitivity of AQP3. amino acids as and also Ni2+ G. Eur. J. 2001; 268: PubMed Scopus Google Scholar). of the examined extracellular to be important for the regulation of AQP3 by residues are not present in the extracellular loops of human AQP3. Ser152 was the in the extracellular loops of AQP3 that was also to be for Ni2+ sensitivity. as well as Tyr124, was the effect of Ni2+ was of His241, Trp128, or Ser152 to of Ni2+ sensitivity. that amino acids are for Ni2+ binding to human AQP3. of to and of His154 to the water permeability of AQP3. We that the of His154 may in important for the of the water address this we with the aromatic amino We that but not had the same water permeability as wild-type AQP3. Human AQP3 was pH-sensitive in the from pH to The of is the amino side chain this pH pH the is and at pH is from the first extracellular and His154 from the second extracellular were to in the regulation of the water permeability by pH. of Ser152 was also with a of AQP3 pH sensitivity. is a common of in a of that have an known as the (reviewed in Ref. Biochemistry. PubMed Scopus Google Scholar). We in AQP3, Ser152 may a to that in the of has been shown to interact with in the of D.A. Biochemistry. 1993; 32: PubMed Scopus Google Scholar). Cells had lower water permeability than cells wild-type AQP3. is that the aromatic side chain of may in with or other aromatic of AQP3. The that AQP3 water permeability is is in line with by and Klaerke (11Zeuthen T. Klaerke D.A. J. Biol. Chem. 1999; 274: 21631-21636Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). other mammalian AQPs have been to be which is expressed in the J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and which is expressed in cells of M. A. Nielsen S. Guggino W.B. Agre P. 1999; PubMed Scopus Google Scholar). In to AQP3, the water permeability for and is at pH and at acidic pH. in the first extracellular was identified as a determinant of the pH sensitivity of residues the pH sensitivity of were not has been to the molecular determinants of AQP3 pH sensitivity. of the protein that amino residues involved in the regulation of AQP3 by Ni2+ or pH are in AQP4 and AQP5. AQP4 AQP5 was or Nickel and acidic pH be used as to the of AQP3 on one and AQP4 and AQP5 on the was performed on a bronchial epithelial cell line from the human Nickel is widely used in modern and inhalation is the primary route of occupational nickel of nickel have been to to lung and lung (reviewed in Ref. 6Kelleher P. Pacheco K. Newman L.S. Environ. Health. Perspect. 2000; 108: 685-696Crossref PubMed Scopus (151) Google Scholar). of nickel with AQP3 to one or of AQP3 AQP4 and AQP5, expressed in the airway have of lung for a but of airway A.S. Y. Am. J. Physiol. 2000; PubMed Google Scholar, Y. S. B. Verkman A.S. J. Physiol. 2001; PubMed Scopus Google Scholar). AQP4 and AQP5 also have of lung has the question of whether lung AQPs are important for airway to be important with to the lung of AQPs and with to AQP3 AQP3 is present in the apical of bronchial epithelial cells in the human but not in the lung (8Nielsen S. King L.S. Christensen B.M. Agre P. Am. J. Physiol. 1997; 273: C1549-C1561Crossref PubMed Google Scholar, 9Matsuzaki T. Suzuki T. Koyama H. Tanaka S. Takata K. J. Histochem. Cytochem. 1999; 47: 1275-1286Crossref PubMed Scopus (143) Google Scholar, 10Kreda S.M. Gynn M.C. Fenstermacher D.A. Boucher R.C. Gabriel S.E. Am. J. Respir. Cell Mol. Biol. 2001; 24: 224-234Crossref PubMed Scopus (218) Google Scholar). the extracellular loops of AQP3 in the human the airway Here, we that Ni2+ to the extracellular loops of human AQP3 and that AQP3 is to extracellular pH. have shown that the epithelial which is for apical into the airway is also S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are with the that the in human lung caused by nickel are to the effect of Ni2+ on apical water and ion The nickel and pH in this The effect of Ni2+ was at acidic and neutral pH than at alkaline pH. may be important to that the airway is alkaline in the of nickel The pH of human lung AQP3 may be of The pH of airway is and to in S. Y. Verkman A.S. J. 2001; PubMed Scopus Google Scholar, S. Y. Verkman A.S. Am. J. Physiol. 2001; PubMed Google Scholar). In a in which human airway epithelial cells were was that the of cells causes in H. B. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). In line with was that the pH of airway is lower in with than in K. R. A. B. Am. J. Respir. 2000; PubMed Scopus Google Scholar). In our AQP3 water permeability decreased when the pH is that airway pH may be and that of the results in with and results in of airway H. B. Am. J. Physiol. 2002; PubMed Scopus Google Scholar, J. 1993; PubMed Scopus Google Scholar, A. J. Physiol. 1999; PubMed Scopus Google Scholar). AQP3 is expressed in several other the and and T. Suzuki T. Koyama H. Tanaka S. Takata K. J. Histochem. Cytochem. 1999; 47: 1275-1286Crossref PubMed Scopus (143) Google Scholar, J. G. M. Nielsen S. Am. J. Physiol. Google Scholar, M. T. B. Verkman A.S. 2000; PubMed Scopus Google Scholar, G. T. Nielsen S. M.C. Am. J. Physiol. 2001; Google Scholar). The most in AQP3 is a in in T. Y. B. A. Verkman A.S. S. A. 2000; PubMed Scopus Google Scholar). be important to the pH and Nickel is one of the most common causes of (6Kelleher P. Pacheco K. Newman L.S. Environ. Health. Perspect. 2000; 108: 685-696Crossref PubMed Scopus (151) Google Scholar, T. S. B. J. 2001; PubMed Scopus Google Scholar). Nickel inhibition of AQP3 may be a to this AQP3 and of the aquaporin have recently been in cells, which an important in the S. H. J. 1999; Full Text PDF PubMed Scopus Google Scholar, A. A. J. 2000; PubMed Scopus Google Scholar). which is well known of AQPs (2Preston G.M. Jung J.S. Guggino W.B. Agre P. J. Biol. Chem. 1993; 268: 17-20Abstract Full Text PDF PubMed Google Scholar, 3Zhang R. van Hoek A.N. Biwersi J. Verkman A.S. Biochemistry. 1993; 32: 2938-2941Crossref PubMed Scopus (126) Google Scholar, 4Kuwahara M. Gu Y. Ishibashi K. Marumo F. Sasaki S. Biochemistry. 1997; 36: 13973-13978Crossref PubMed Scopus (91) Google Scholar) and a cause of (6Kelleher P. Pacheco K. Newman L.S. Environ. Health. Perspect. 2000; 108: 685-696Crossref PubMed Scopus (151) Google Scholar, G. Am. J. 2002; PubMed Scopus Google Scholar), inhibits the cell in cells A. A. J. 2000; PubMed Scopus Google Scholar). the that nickel the water permeability of AQP3, is to that of regulation of cells is an important to nickel and to the component of nickel pneumoconiosis.
Zelenina et al. (Fri,) studied this question.