Quaternary Ammonium Compounds as Water Channel Blockers
2006; Elsevier BV; Volume: 281; Issue: 20 Linguagem: Inglês
10.1074/jbc.m513072200
ISSN1083-351X
AutoresFrank Detmers, Bert L. de Groot, E. Matthias Müller, Andrew Hinton, Irene B. M. Konings, Mozes Sze, Sabine L. Flitsch, Helmut Grubmüller, Peter M.T. Deen,
Tópico(s)Membrane-based Ion Separation Techniques
ResumoExcessive water uptake through Aquaporins (AQP) can be life-threatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC50 values of 1.4, 6.2, and 9.8 μm, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp128) and E (Asp185) loop, and the A(Tyr37-Asn42-Thr44) loop of the neighboring monomer, but not directly with Tyr186. The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound. Excessive water uptake through Aquaporins (AQP) can be life-threatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC50 values of 1.4, 6.2, and 9.8 μm, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp128) and E (Asp185) loop, and the A(Tyr37-Asn42-Thr44) loop of the neighboring monomer, but not directly with Tyr186. The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound. Aquaporins form a large family of integral membrane proteins that facilitate specific, efficient, and passive permeation of water, whereas members of the aqua(glycero)porins also permeate small solutes, such as glycerol and urea (1Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1029) Google Scholar). In mammals, 13 different aqua(glycero)porins have been identified, which differ in their tissues of expression, regulation, and selectivity. The gross structure of these membrane proteins is conserved and consists of six transmembrane domains with cytoplasmic N and C termini. The 1st intracellular (B) and 2nd extracellular (E) loop, both containing the highly conserved Asn-Pro-Ala (NPA) motive, fold back into the membrane and form the central part of the water pore (2Murata K. Mitsuoka K. Hirai T. Walz T. Agre P. Heymann J.B. Engel A. Fujiyoshi Y. Nature. 2000; 407: 599-605Crossref PubMed Scopus (1434) Google Scholar, 3Jung J.S. Preston G.M. Smith B.L. Guggino W.B. Agre P. J. Biol. Chem. 1994; 269: 14648-14654Abstract Full Text PDF PubMed Google Scholar). Although every monomer is a functional water pore, aquaporins are expressed as homotetramers (4Van Hoek A.N. Hom M.L. Luthjens L.H. de Jong M.D. Dempster J.A. van Os C.H. J. Biol. Chem. 1991; 266: 16633-16635Abstract Full Text PDF PubMed Google Scholar, 5Smith B.L. Agre P. J. Biol. Chem. 1991; 266: 6407-6415Abstract Full Text PDF PubMed Google Scholar). The atomic structures of human AQP1 3The abbreviations used are: AQP, aquaporin; MD, molecular dynamics; TEA, tetraethylammonium; TMA, tetramethyl; WT, wild type; ER, endoplasmic reticulum. 3The abbreviations used are: AQP, aquaporin; MD, molecular dynamics; TEA, tetraethylammonium; TMA, tetramethyl; WT, wild type; ER, endoplasmic reticulum. (2Murata K. Mitsuoka K. Hirai T. Walz T. Agre P. Heymann J.B. Engel A. Fujiyoshi Y. Nature. 2000; 407: 599-605Crossref PubMed Scopus (1434) Google Scholar) and GlpF from Eschericia coli (6Fu D. Libson A. Miercke L.J. Weitzman C. Nollert P. Krucinski J. Stroud R.M. Science. 2000; 290: 481-486Crossref PubMed Scopus (881) Google Scholar), and real time molecular dynamics (MD) studies of these proteins (7de Groot B.L. Grubmuller H. Science. 2001; 294: 2353-2357Crossref PubMed Scopus (820) Google Scholar, 8Tajkhorshid E. Nollert P. Jensen M.O. Miercke L.J. O'Connell J. Stroud R.M. Schulten K. Science. 2002; 296: 525-530Crossref PubMed Scopus (764) Google Scholar) established the molecular mechanism of water permeation. Aquaporins are involved in the regulation of the water balance in many tissues (1Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1029) Google Scholar, 9van Os C.H. Kamsteeg E.J. Marr N. Deen P. M.T. Pflugers Arch. 2000; 440: 513-520Crossref PubMed Google Scholar). In the kidney, AQP1 is present in the proximal tubules and thin descending limbs of Henle, in which almost 90% of the 180 liters of daily formed pro-urine is reabsorbed. The remaining volume is concentrated via the vasopressin-regulated AQP2, which is present in the apical membrane of principal cells of the renal collecting duct (10Deen P.M.T. Verdijk M.A.J. Knoers N.V.A.M. Wieringa B. Monnens L.A.H. van Os C.H. van Oost B.A. Science. 1994; 264: 92-95Crossref PubMed Scopus (762) Google Scholar, 11Fushimi K. Uchida S. Hara Y. Hirata Y. Marumo F. Sasaki S. Nature. 1993; 361: 549-552Crossref PubMed Scopus (868) Google Scholar), whereas AQP3 and AQP4, which are present in the basolateral membrane of these cells, form the exit pathway of water to the interstitium (1Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1029) Google Scholar, 12Deen P.M.T. Van Balkom B.W.M. Kamsteeg E.J. Eur. J. Cell Biol. 2000; 79: 523-530Crossref PubMed Scopus (46) Google Scholar). Besides the kidney, AQP3 is also found in the gastrointestinal tract and the stratum corneum of the skin, where it exhibits a high water permeability (13Sougrat R. Morand M. Gondran C. Barre P. Gobin R. Bonte F. Dumas M. Verbavatz J.M. J. Investig. Dermatol. 2002; 118: 678-685Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The clinical importance of AQPs is shown by their role in several disturbed water balance disorders, which can severely affect the quality of life and can be life threatening. The lack of AQP2 in states of excessive water loss is fundamental to widely occurring nephrogenic diabetes insipidus, a disease in which the kidney is unable to concentrate urine in response to vasopressin (1Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1029) Google Scholar, 14Knoers N.V.A.M. Deen P.M.T. Pediatr. Nephrol. 2001; 16: 1146-1152Crossref PubMed Scopus (93) Google Scholar). In mice, lack of AQP3 also results in an nephrogenic diabetes insipidus phenotype, indicating that AQP3 might also have an important role in urine concentration (15Ma T. Song Y. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4386-4391Crossref PubMed Scopus (337) Google Scholar). AQP5 is present in the lung and exocrine glands, such as salivary and sweat glands (16Song Y. Sonawane N. Verkman A.S. J. Physiol. 2002; 541: 561-568Crossref PubMed Scopus (88) Google Scholar), and the reduced saliva production in AQP5 knock-out mice underscores the important role of AQP5 in this process (17Ma T. Song Y. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Biol. Chem. 1999; 274: 20071-20074Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Paradoxically, disorders characterized by excessive water transport have also shown or have been ascribed to AQPs. AQP1 has been shown to be involved in tumor growth (18Saadoun S. Papadopoulos M.C. Hara-chikuma M. Verkman A.S. Nature. 2005; 434: 786-792Crossref PubMed Scopus (646) Google Scholar), and suggested to have a role in the development of pulmonary edema, eye glaucoma, and cyst formation in polycystic kidney disease (19King L.S. Agre P. Annu. Rev. Physiol. 1996; 58: 619-648Crossref PubMed Scopus (458) Google Scholar). In several disorders, excessive renal water uptake is due to high renal plasma membrane AQP2 expression, which can lead to life threatening hyponatremia (20Pedersen R.S. Bentzen H. Bech J.N. Nyvad O. Pedersen E.B. Kidney Int. 2003; 63: 1417-1425Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). AQP4 is, among other tissues, expressed in cells lining brain ventricles and has been shown to have a key role in the formation of brain edema (21Manley G.T. Fujimura M. Ma T. Noshita N. Filiz F. Bollen A.W. Chan P. Verkman A.S. Nat. Med. 2000; 6: 159-163Crossref PubMed Scopus (1298) Google Scholar), as a consequence of hyponatremia, stroke, accidents, and cancer. Considering the important roles of AQPs in excessive water transport, reversible aquaporin-specific blockers are clinically highly desirable. Such blockers for AQP1, AQP2, and AQP3 could serve well as diuretics and anti-tumor drugs (AQP1), whereas a timely administration of AQP4-specific blockers might reduce the formation of brain edema. Such blockers, however, are yet not available. Mercury, silver, and gold have been shown to inhibit AQPs (22Preston G.M. Jung J.S. Guggino W.B. Agre P. J. Biol. Chem. 1993; 268: 17-20Abstract Full Text PDF PubMed Google Scholar, 23Niemietz C.M. Tyerman S.D. FEBS Lett. 2002; 531: 443-447Crossref PubMed Scopus (245) Google Scholar), but these metals are highly toxic and their inhibition is not reversible. Recently, however, it was reported that the ion channel blocker tetraethylammonium (TEA) weakly inhibits AQP1-mediated water permeability in oocytes (24Brooks H.L. Regan J.W. Yool A.J. Mol. Pharmacol. 2000; 57: 1021-1026PubMed Google Scholar) and mammalian cells (25Yool A.J. Brokl O.H. Pannabecker T.L. Dantzler W.H. Stamer W.D. BMC Physiol. 2002; 2: 4Crossref PubMed Google Scholar). Because TEA can be chemically modified, it is potentially a promising lead compound for the development of AQP-specific and selective blockers. Therefore, we here tested its selectivity toward different water channels, determined its potency of inhibition on different AQPs, and determined the TEA-active site in AQP1, using a combination of an oocyte swelling assay, molecular docking, and MD simulations. Constructs—Expression constructs encoding human (h) AQP1 (pXβg-ev1hAQP1; kind gift of Peter Agre (3Jung J.S. Preston G.M. Smith B.L. Guggino W.B. Agre P. J. Biol. Chem. 1994; 269: 14648-14654Abstract Full Text PDF PubMed Google Scholar)), pT7TshAQP2 (10Deen P.M.T. Verdijk M.A.J. Knoers N.V.A.M. Wieringa B. Monnens L.A.H. van Os C.H. van Oost B.A. Science. 1994; 264: 92-95Crossref PubMed Scopus (762) Google Scholar), or pBSKSIIhAQP4 (26Lu M. Lee M.D. Smith B.L. Jung J.S. Agre P. Verdijk M.A.J. Merkx G. Rijss J.P.L. Deen P.M.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10908-10912Crossref PubMed Scopus (177) Google Scholar) were previously described. To generate pT7Ts-hAQP3, a 1444-bp BamHI-EcoRV fragment from pBSKSII hAQP3 (kind gift of Kenneth Ishibashi (27Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi Y. Gojobori T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (532) Google Scholar)) was cloned into the BglII/EcoRV sites of the oocyte expression vector pT7Ts (10Deen P.M.T. Verdijk M.A.J. Knoers N.V.A.M. Wieringa B. Monnens L.A.H. van Os C.H. van Oost B.A. Science. 1994; 264: 92-95Crossref PubMed Scopus (762) Google Scholar). To obtain pT7Ts-hAQP5, an 887-bp BamHI/EcoRV fragment was cut from pBSKSII hAQP5 (kindly provided by Peter Agre (28Lee M.D. Bhakta K.Y. Raina S. Yonescu R. Griffin C.A. Copeland N.G. Gilbert D.J. Jenkins N.A. Preston G.M. Agre P. J. Biol. Chem. 1996; 271: 8599-8604Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)) and cloned into the BglII/EcoRV sites of pT7Ts. With the QuikChange site-directed mutagenesis kit (Stratagene, Heidelberg, Germany), mutations were introduced into the human AQP1-5 cDNA sequences, using the following forward primers: CTGGGCTTTAAATTCCCGGTG for Y37F, GGGAACGCCCAGACGGCGGTGCAGGAC for N42A, CAACCAGGCGGCGGTGCAGGAC for T44A, CGCAATTCCCTGGCTGATGGTGTCAAC for D128S, GCTATTTCCTACACCGGTTGTGGG for D185S, and GCTATTGACTTCACCGGTTGTGGGATTAACC for Y186F in AQP1; CCACCTCCTCGGGATCCATTTCACCGGCTGC for Y178F in AQP2; CATTGGCACCTCAATGGGCTTCTACTCCGGCTATGC for N209Y in AQP3; GCAATCAATTTTACCGGTGCCAGC for Y185F in AQP4; GGAATCTACTACACCGGTTGCTCCATGAAC for F179Y in AQP5; and GTCGGAATCTTCTACACCGGTTGCTCCATGAAC for Y178F/F179Y in AQP5. With these primers, restriction sites were introduced (DraI (Y37F), BsgI (N42A/T44A), HincII (D128S), and AgeI in AQP4-Y185F, AQP5-F179Y, and AQP5-Y178F/F179Y) or deleted (BstXI for AQP2-Y178F, NcoI site for AQP3-N209Y). Sequence analysis of selected clones confirmed that only the desired mutations were introduced. Water Permeability Measurements—Expression constructs were linearized with SalI (pT7TshAQP2, pT7TshAQP5), XbaI (pT7TshAQP3, pBSKSIIhAQP4), or PstI (pXβg-ev1hAQP1). G-capped cRNA transcripts were synthesized in vitro using T7 RNA polymerase (AQP2, -3, and -5) or T3 RNA polymerase (AQP1 and -4). Transcription, and the isolation, integrity checks, and determination of the concentration of the cRNAs was done as described (29Deen P.M.T. Croes H. van Aubel R.A. Ginsel L.A. van Os C.H. J. Clin. Investig. 1995; 95: 2291-2296Crossref PubMed Scopus (218) Google Scholar). Xenopus oocytes were isolated and stored as described (30Mulders S.M. Bichet D.G. Rijss J.P.L. Kamsteeg E.J. Arthus M.F. Lonergan M. Fujiwara M. Morgan K. Leijendekker R. van der Sluijs P. van Os C.H. Deen P. M.T. J. Clin. Investig. 1998; 102: 57-66Crossref PubMed Scopus (224) Google Scholar). The oocytes were injected with 0.2-0.5 ng of cRNA coding for AQP1-5 and 0.5-1.0 ng of cRNA for the described mutants. One day after injection the follicular membranes were removed, and 2 days after injection the water permeability (Pf ± S.E.; N ≧ 8; in μm/s) was measured using a standard swelling assay (10Deen P.M.T. Verdijk M.A.J. Knoers N.V.A.M. Wieringa B. Monnens L.A.H. van Os C.H. van Oost B.A. Science. 1994; 264: 92-95Crossref PubMed Scopus (762) Google Scholar), except that here ND96P saline buffer (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 2.5 mm sodium pyruvate, 5 mm HEPES, pH 7.6, 200 mosm ND96P) instead of modified Barth's solution was used, because the tested inhibitors were better soluble in ND96P. For the swelling assays, ND96P was diluted 10 times with Milli-Q water. To test TEA-like compounds for their inhibitory activity, injected oocytes were pretreated with the compound in ND96P for 15 min after which they were subjected to a swelling assay in 20 mosm ND96P in the presence of the compound. Similar results were obtained without the preincubation step. The data shown are an average of five experiments (with 5 different batches of oocytes) in which 10 to 12 oocytes were tested at every condition. The Pf values under different conditions were compared within one batch of oocytes. The measured Pf values were statistically analyzed in an unpaired Student's t test. p values < 0.05 were considered significantly different. For the calculation of the IC50 values of potential blockers, the ratio of inhibition (I/I0) was determined, in which “I” is the Pf at a certain inhibitor concentration and I0 is the Pf in the absence of inhibitor. Both values are corrected for the Pf of non-injected oocytes. The I/I0 values of each individual experiment were plotted against corresponding TEA concentrations and subjected to the automatic curve-fitting procedure in Microsoft Excel. The IC50 ± S.E. values were calculated from four independent experiments. Membrane Isolation and Immunoblotting—Total membranes and plasma membranes were isolated from at least 8 oocytes per sample as described (31Kamsteeg E.J. Deen P.M.T. Biochem. Biophys. Res. Commun. 2001; 282: 683-690Crossref PubMed Scopus (45) Google Scholar). Protein samples were denatured by incubation for 30 min at 37 °C in Laemmli buffer and blotted as described (29Deen P.M.T. Croes H. van Aubel R.A. Ginsel L.A. van Os C.H. J. Clin. Investig. 1995; 95: 2291-2296Crossref PubMed Scopus (218) Google Scholar). Next, the blots were incubated overnight with 1:600 diluted mouse α-AQP1 (32Deen P.M.T. Nielsen S. Bindels R.J.M. van Os C.H. Pflugers Arch. 1997; 433: 780-787Crossref PubMed Scopus (45) Google Scholar), 1:1500 diluted rabbit α-AQP2 (29Deen P.M.T. Croes H. van Aubel R.A. Ginsel L.A. van Os C.H. J. Clin. Investig. 1995; 95: 2291-2296Crossref PubMed Scopus (218) Google Scholar), or 1:500 diluted α-AQP4 (33Van Balkom B.W.M. Van Raak M. Breton S. Pastor-Soler N. Bouley R. van der Sluijs P. Brown D. Deen P. M.T. J. Biol. Chem. 2003; 278: 1101-1107Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) in TBST buffer (20 mm Tris, 140 mm NaCl, 0.1% Tween, pH 7.6) supplemented with 1% nonfat dried milk. As secondary antibodies, a 1:5000 dilution of goat α-rabbit IgG (Sigma) for AQP2 and AQP4, or a 1:2000 dilution of sheep α-mouse IgG (Sigma) for AQP1, both coupled to horseradish peroxidase, were used. Finally, AQP proteins were visualized using enhanced chemiluminescence (Pierce). Determination of TEA Docking Sites—The molecular docking package DOCK 4.0 (34Ewing T.J. Kuntz I.D. J. Comp. Chem. 1997; 18: 1175-1189Crossref Scopus (548) Google Scholar) was used to dock TEA into the x-ray structure of bovine AQP1 (Protein Data Bank code 1J4N). Atomic coordinates including hydrogen atoms and charge information for TEA were generated using the SYBYL package from Tripos Inc. (SYBYL 6.9, Tripos Inc., St. Louis, MI). A three-dimensional model of TEA was build using the CONCORD (57Pearlman R.S. CONCORD User's Manual. Tripos Inc., St. Louis, MO1998Google Scholar) package. Using the SPHGEN program (35Kuntz I.D. Blaney J.M. Oatley S.J. Langridge R. Ferrin T.E. J. Mol. Biol. 1982; 161: 269-288Crossref PubMed Scopus (1873) Google Scholar), clusters of overlapping spheres were created in the upper vestibule of bovine AQP1. The original SPHGEN output file was edited using the InsightII package (58Inc Accelrys InsightII Modeling Environment, Release 2000. Accelrys Inc., San Diego, CA2000Google Scholar) until a cluster of 16 spheres remained that filled the solvent accessible surface area of AQP1. Hydrogens were added to the protein and charged using a Gasteiger-Hückel potential. The Dock package flexibly orientated the TEA molecule within the defined docking region, scoring and ranking the 100 best orientations. Orientations were ranked using the energy score of DOCK. The molecular visualization package WITNOTP (59Widmer A. WITNOTP: A Computer Program for Molecular Modeling. Novartis, Basel, Switzerland1997Google Scholar) was used to inspect TEA in the upper vestibule of AQP1. Four representative orientations of TEA were selected as starting positions for subsequent MD studies. Molecular Dynamics Simulations—MD simulations were started from the bovine AQP1 x-ray structure (PDB code 1J4N (36Sui H. Han B.G. Lee J.K. Walian P. Jap B.K. Nature. 2001; 414: 872-878Crossref PubMed Scopus (961) Google Scholar)), after modifying the structure to adopt the sequence of human AQP1. The 21 mutations and the insertion of 2 residues were carried out using the WHAT IF package (37Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3363) Google Scholar). The percentage of sequence identity is 90.63%. None of the mutations or insertions is located in the pore region. AQP1 was simulated as a tetramer embedded in a solvated palmitoyloleoyl phosphatidylethanolamine lipid bilayer. The simulation system contained 8,340 protein atoms, 14,093 lipid atoms, 19,769 SPC water molecules (38Berendsen H.J. Postma J.P. van Gunsteren W.F. Hermans J. Pullman B. Intermolecular Forces. D. Reidel Publishing Company, Dordrecht2004Google Scholar), and four chloride ions, resulting in a system size of 81,739 atoms. The simulation setup and conditions were identical to those described before (7de Groot B.L. Grubmuller H. Science. 2001; 294: 2353-2357Crossref PubMed Scopus (820) Google Scholar). In short, MD simulations were carried out using the Gromacs simulation suite (39Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). Lincs and Settle (40Hess B. Bekker H. Berendsen H.J. Fraaije J.G. J. Comp. Chem. 1997; 18: 1463-1472Crossref Scopus (11574) Google Scholar, 41Miyamoto S. Kollman P.A. J. Comp. Chem. 1992; 13: 952-962Crossref Scopus (5201) Google Scholar) were applied to constrain covalent bond lengths, allowing an integration time step of 2 fs. Electrostatic interactions were calculated using the Particle-Mesh Ewald method (40Hess B. Bekker H. Berendsen H.J. Fraaije J.G. J. Comp. Chem. 1997; 18: 1463-1472Crossref Scopus (11574) Google Scholar). The temperature was kept constant by separately coupling (τ = 0.1 ps) the protein, lipids, and solvent to an external temperature bath (42Berendsen H.J. Postma J.P. DiNola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23458) Google Scholar). The pressure was kept constant by weak coupling (τ = 1.0 ps) in the z-direction (normal to the bilayer plane) to a pressure bath (42Berendsen H.J. Postma J.P. DiNola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23458) Google Scholar). The gromacs force field was applied, which is the gromos 87 force field (43van Gunsteren W.F. Berendsen H.J. GROMOS Manual, BIOMOS. Biomolecular Software, Laboratory of Physical Chemistry, University of Groningen, The Netherlands1987Google Scholar) with slight modifications (44Van Buuren A.R. Marrink S.-J. Berendsen H. J.C. J. Phys. Chem. 1993; 97: 9206-9212Crossref Scopus (380) Google Scholar) and explicit hydrogens on aromatic residues. To equilibrate the system, 500 ps of MD were performed with harmonic position restraints on all non-hydrogen protein atoms (k = 1000 kJ/(mol nm)). All subsequent simulations started from the resulting structure. In total, seven simulations were performed, five of the wild type protein (WT), and two of the Y186F mutant. The simulation length of the seven production runs was 15 ns for each of the simulations, totaling 105 ns of simulation time. For the simulations including TEA and tetramethyl (TMA), initial positions for the inhibitors were derived from the docking approach described above. For WT_TEA1, TEA was placed at four different docking positions within the four monomeric channels of the tetramer, respectively (for the selection procedure of the docking orientations, see above). After inserting the TEA molecules, those water molecules that showed significant overlap with the inhibitor were removed from the simulation system. Because in this simulation TEA remained stably bound to the protein only in one of the four positions (see also “Results”), a second simulation (WT_TEA2) was started with TEA molecules bound to this position in all four monomers. In both simulations, to re-equilibrate the system, the position of the central nitrogen atom of the TEA was kept fixed by a harmonic positional restraint (k = 10000 kJ/(mol nm)) for 500 ps. The third simulation including TEA (WT_TEA3) started from the same conformation as WT_TEA2, but the equilibration period was extended to 3 ns before the production phase started. The WT_TMA simulation started, like the WT_TEA1 simulation, with TMA bound to four different positions obtained from the docking study described above. To all simulation systems including TEA and TMA, four additional chloride ions were added to compensate for the net charge of the quaternary ammonium ions. The starting structures for the simulations of the Y186F mutant, Y186F_free and Y186F_TEA, were modeled from the structures, after equilibration, of the WT_free and WT_TEA3 simulation, respectively, by replacing the tyrosine hydroxide groups by a proton. Free energy changes associated with the Y186F mutation were estimated with thermodynamic integration calculations. The difference in stability between the WT and mutant protein was estimated from the free energy difference between the mutation in the folded protein and in a model of the unfolded state (modeled as a tripeptide in solution). All thermodynamic integration simulations were carried out using the method of slow growth (45Bash P.A. Singh U.C. Langridge R. Kollman P.A. Science. 1987; 236: 564-568Crossref PubMed Scopus (423) Google Scholar), i.e. by gradually introducing the mutation into the simulated system (using soft-core parameters α = 0 (resulting in linear interpolation of the non-bonded interactions) and σ = 0.3 nm) during a simulation period of 1 ns. For both the folded state and unfolded state (tripeptide), forward and backward mutations were simulated to ensure that the mutation was sufficiently reversible and hysteresis effects were small. For molecular visualization the pymol program (46DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar) was used. Quaternary Ammonium Compounds Tested on Human AQP1—Brooks et al. (24Brooks H.L. Regan J.W. Yool A.J. Mol. Pharmacol. 2000; 57: 1021-1026PubMed Google Scholar) recently reported that 100 μm TEA could reversibly inhibit water permeation through AQP1. To test whether we could reproduce these data, Xenopus oocytes were injected with 0.2 ng of AQP1 cRNA and tested in an oocyte-swelling assay in the presence or absence of 100 μm TEA. Oocytes treated with TEA showed a reduced Pf of 44 ± 14% compared with controls (Fig. 1A). After subjection to a swelling assay in the presence of TEA, oocytes were washed four times and allowed to recover to the normal volume for 4 h. Subsequent analysis of these oocytes in a swelling assay in the absence of TEA (Fig. 1A, wash) revealed a Pf that was significantly higher than when they were treated with TEA (p = 0.038), and which was not significantly different from AQP1-expressing control oocytes (p = 0.074). These data confirm that the water permeation through AQP1 can be reversibly inhibited by TEA. To determine whether the lengths of the carbon side chain of quaternary ammonium compounds affected the inhibition of AQP1 water permeability, 100 μm concentrations of TMA-, tetrapropyl-(TPrA), tetrabutyl-(TBA), and tetrapentyl (TPeA) ammonium compounds were tested on AQP1-expressing oocytes. Determination of the Pfs revealed that, besides TEA, only TPrA significantly inhibited the AQP1 water permeability (32 ± 15%; p = 0.027; Fig. 1B). The inhibition by TPrA was not significantly different from that of TEA and was thus as effective as TEA at the tested concentration of 100 μm. In principle, the effect of TEA and TPrA on AQP1 water permeation could be either a direct effect of the compound on AQP1 functioning or a result of a reduced plasma membrane expression of AQP1 in the presence of the blockers. To address this question, AQP1-expressing oocytes were incubated with 100 μm TMA, TEA, or TPrA for 15 min, after which plasma membranes were isolated from eight oocytes in duplicate and immunoblotted for AQP1 (Fig. 1C). Analysis of the immunoblot signals revealed similar plasma membrane expression of AQP1 for oocytes treated with TEA/TPrA versus TMA (p = 0.11/0.20 respectively; n = 4). As differences in AQP1 amounts revealed different signal intensities at this exposure time (dilution series), this indicated that the reduced water permeability with TEA or TPrA was due to a direct inhibition of AQP1 instead of effects on the levels of AQP1 plasma membrane expression. Screening of AQP2-5 for Inhibitory Effects of Quaternary Ammonium Compounds—To determine whether the quaternary ammonium compounds specifically inhibit AQP1 or also affect the water permeation of other AQPs, oocytes expressing human AQP2, AQP3, AQP4, or AQP5 were subjected to swelling assays in the presence or absence of 100 μm TMA, TEA, TPrA, TBA, or TPeA. Determination of the Pfs revealed that, besides AQP1, the water permeability of AQP2 and AQP4 was inhibited by TEA to 49 ± 15 and 55 ± 18%, respectively (data not shown). In addition, the other four ammonium compounds did not have a significant inhibitory effect on the water permeability of AQP2 or AQP4, and the water permeability of AQP3 or AQP5 was not inhibited by any of the
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