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• W2283938713 abstract "The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family. The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family. The aquaporin (AQP) 3The abbreviations used are: AQPaquaporinFRETForster resonant energy transferFRAPfluorescence recovery after photobleachingTMtransmembrane domainMDCKMadin-Darby canine kidney cellsBN-PAGEblue native-PAGEbis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineANOVAanalysis of variance. family of integral membrane proteins facilitate both osmosis and diffusion of small polar molecules through biological membranes. A wealth of medium-to-high resolution structural data for various family members (there are 48 AQP structures in the Protein Data Bank) all suggest that the AQP homotetrameric quaternary structure is highly conserved despite diversity in solute permeability, subcellular localization, and trafficking responses of individual AQPs. Early biochemical work, using carboxyl-amine fusion dimers (consisting of 1 wild-type unit and 1 unit lacking the cysteine residue required for mercurial inhibition), showed that monomers are the functional AQP units (1.Jung J.S. Preston G.M. Smith B.L. Guggino W.B. Agre P. Molecular structure of the water channel through aquaporin CHIP: the hourglass model.J. Biol. Chem. 1994; 269: 14648-14654Abstract Full Text PDF PubMed Google Scholar); numerous molecular dynamics simulation studies support this (2.Hub J.S. Grubmuller H. de Groot B.L. Dynamics and energetics of permeation through aquaporins: what do we learn from molecular dynamics simulations?.Handb. Exp. Pharmacol. 2009; 2009: 57-76Crossref Scopus (86) Google Scholar). Recent work has suggested that isolated AQP monomers are equally capable of facilitating water transport as those incorporated into a tetramer (3.Horner A. Zocher F. Preiner J. Ollinger N. Siligan C. Akimov S.A. Pohl P. The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues.Sci. Adv. 2015; 1: e1400083Crossref PubMed Scopus (101) Google Scholar). Therefore it is not clear why AQPs retain this tetrameric structure. Regulation of AQP function by the formation of heterotetramers has been suggested for some plant AQPs (4.Chevalier A.S. Chaumont F. Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.Plant Cell Physiol. 2015; 56: 819-829Crossref PubMed Scopus (52) Google Scholar). The fifth, central pore formed at the 4-fold axis of the tetramer has also been suggested to transport carbon dioxide (5.Kaldenhoff R. Kai L. Uehlein N. Aquaporins and membrane diffusion of CO2 in living organisms.Biochim. Biophys. Acta. 2014; 1840: 1592-1595Crossref PubMed Scopus (75) Google Scholar) and cations (6.Yu J. Yool A.J. Schulten K. Tajkhorshid E. Mechanism of gating and ion conductivity of a possible tetrameric pore in aquaporin-1.Structure. 2006; 14: 1411-1423Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), at least in mammalian AQP1. Trigger-induced relocalization of AQP-containing vesicles to the plasma membrane is a well established regulatory mechanism for AQPs; the best studied example of this is relocalization of AQP2 to the apical membrane of the collecting duct in the mammalian kidney in response to arginine vasopressin (also called anti-diuretic hormone). There is also evidence for similar mechanisms for other AQPs including AQP1 (7.Conner M.T. Conner A.C. Bland C.E. Taylor L.H. Brown J.E. Parri H.R. Bill R.M. Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel.J. Biol. Chem. 2012; 287: 11516-11525Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), AQP5 (8.Cho G. Bragiel A.M. Wang D. Pieczonka T.D. Skowronski M.T. Shono M. Nielsen S. Ishikawa Y. Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.Biochim. Biophys. Acta. 2015; 1850: 784-793Crossref PubMed Scopus (16) Google Scholar), and AQP7 (9.Kishida K. Kuriyama H. Funahashi T. Shimomura I. Kihara S. Ouchi N. Nishida M. Nishizawa H. Matsuda M. Takahashi M. Hotta K. Nakamura T. Yamashita S. Tochino Y. Matsuzawa Y. Aquaporin adipose, a putative glycerol channel in adipocytes.J. Biol. Chem. 2000; 275: 20896-20902Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). A naturally occurring AQP2 mutant (R187C) has also been reported that is unable to relocalize in response to arginine vasopressin or to form tetramers (10.Kamsteeg E.J. Wormhoudt T.A. Rijss J.P. van Os C.H. Deen P.M. An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus.EMBO J. 1999; 18: 2394-2400Crossref PubMed Scopus (170) Google Scholar). Given the ubiquity of these regulatory responses across the AQP family and the conservation of the tetrameric quaternary structure, it may be that these trigger-induced relocalization responses involve interaction with proteins that only recognize the tetrameric form of AQPs. aquaporin Forster resonant energy transfer fluorescence recovery after photobleaching transmembrane domain Madin-Darby canine kidney cells blue native-PAGE 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine analysis of variance. Here we demonstrate that intracellular loop D of AQP4 forms vital homomeric interactions between AQP subunits that stabilize the tetrameric quaternary structure. We also show that loss of tetramerization does not affect single channel water permeability. Our data suggest that tetramerization is not required for AQP4 to be trafficked through the endoplasmic reticulum and Golgi to the plasma membrane, but that unlike wild-type AQP4, the non-tetrameric mutants are unable to relocalize to the plasma membrane in response to changes in local osmolality. Finally, based on loss and gain of oligomerization mutants of AQP1 and AQP3, we suggest that loop D-mediated inter-monomer interactions may control formation of the signature quaternary structure of the family. Human AQP4 cDNA cloned into pDEST47 (Life Technologies) was used as previously described (11.Kitchen P. Day R.E. Taylor L.H. Salman M.M. Bill R.M. Conner M.T. Conner A.C. Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel.J. Biol. Chem. 2015; 290: 16873-16881Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). An untagged AQP4 construct was created from this by mutagenesis of the first two codons of the GFP linker peptide to stop codons. These were used as templates for mutagenesis following the QuikChange protocol (Stratagene). Mutagenic primers were synthesized by Sigma. Mutant plasmids were amplified in TOP10 Escherichia coli with 100 ng/ml of ampicillin selection. Plasmid DNA was purified using a Wizard Maxiprep kit (Promega) and diluted to 1 mg/ml for transfection. HEK293 cells were cultured routinely in DMEM with l-glutamine (Sigma) supplemented with 10% (v/v) fetal bovine serum (Sigma) and without antibiotics in humidified 5% (v/v) CO2 in air at 37 °C. Cells were seeded into either tissue culture treated 6-well plates (Falcon) for Blue Native (BN)-PAGE and biotinylation or 35-mm FluoroDishesTM (World Precision Instruments) for confocal microscopy. Cells were transfected (at 50% confluence) using polyethyleneimine (branched, average Mr ∼25,000, Sigma) as previously described (11.Kitchen P. Day R.E. Taylor L.H. Salman M.M. Bill R.M. Conner M.T. Conner A.C. Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel.J. Biol. Chem. 2015; 290: 16873-16881Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). MDCK cells were cultured in the same conditions as HEK293 cells. Stable transfections were done using the neomycin resistance gene on the pDEST47 vector. Cells were transiently transfected as described above, then trypsinized and serially diluted into tissue culture plates after 24 h. Cells were treated with 700 μg/ml of G418 antibiotic for 2 weeks, with medium replaced every third day. GFP-expressing resistant colonies were picked using cloning cylinders (Sigma) and serially diluted. The lowest dilution that grew to confluence was used to generate a stable cell line. Cellular protein was subjected to SDS-PAGE and Western blotting as previously described (11.Kitchen P. Day R.E. Taylor L.H. Salman M.M. Bill R.M. Conner M.T. Conner A.C. Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel.J. Biol. Chem. 2015; 290: 16873-16881Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and the highest expressing clone was chosen for experiments. No endogenous AQP4 was detected in Western blots. Reduced G418 pressure (300 μg/ml) was used to maintain the stable cell lines after colony isolation. All cells were routinely tested for mycoplasma using the EZ-PCR test kit (Biological Industries), and all data reported are from cells that tested negative. HEK293 and MDCK cells both expressed only the M1 isoform of AQP4 from the wild-type AQP4 construct. This was confirmed by Western blotting comparing the wild-type construct to AQP4 constructs in which either the M1 or M23 translation initiation sites were removed, as previously described (11.Kitchen P. Day R.E. Taylor L.H. Salman M.M. Bill R.M. Conner M.T. Conner A.C. Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel.J. Biol. Chem. 2015; 290: 16873-16881Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). AQP4-GFP constructs were imaged in live HEK293 cells using a Zeiss LSM 780 confocal microscope with a ×63 1.4 NA oil immersion objective. GFP was excited using the 488-nm line of an argon laser, Venus using the 514-nm line and mTurquoise2 using a 405-nm diode laser. Hypotonic exposure was performed by adding 3 ml of ddH2O to cells in 1 ml of growth medium (280 to 70 mosmol/kg of H2O). Line profiles across the cell membrane and cytoplasm were extracted using ImageJ as previously described (7.Conner M.T. Conner A.C. Bland C.E. Taylor L.H. Brown J.E. Parri H.R. Bill R.M. Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel.J. Biol. Chem. 2012; 287: 11516-11525Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Relative membrane expression was calculated from these profiles (3 profiles per cell and at least 3 cells per image) using in-house Matlab code. Fluorescence recovery after photobleaching (FRAP) was done using a circular bleaching 1-μm area of radius. Recovery curves were fitted to a single phase exponential recovery function and diffusion coefficients were calculated using the approach and equations of Kang et al. (12.Kang M. Day C.A. Kenworthy A.K. DiBenedetto E. Simplified equation to extract diffusion coefficients from confocal FRAP data.Traffic. 2012; 13: 1589-1600Crossref PubMed Scopus (135) Google Scholar). Recovery curves were collected from 5 different cells on the same plate per experiment. FRET experiments were done using the sensitized emission methodology with the FRET signal corrected for donor emission in the acceptor channel and direct excitation of the acceptor, following van Rheenen et al. (13.van Rheenen J. Langeslag M. Jalink K. Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission.Biophys. J. 2004; 86: 2517-2529Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and normalized to the acceptor emission to give an apparent FRET efficiency. The contrast of some images in the figures was adjusted manually using ImageJ to aid the eye. All analysis was performed on raw, unadjusted images. Cell surface amines were biotinylated using an amine-reactive biotinylation reagent that is not cell permeable (Thermo number 21328, EZ-Link Sulfo-NHS-SS-Biotin), and surface AQPs were detected using a neutravidin-based ELISA as previously described (11.Kitchen P. Day R.E. Taylor L.H. Salman M.M. Bill R.M. Conner M.T. Conner A.C. Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel.J. Biol. Chem. 2015; 290: 16873-16881Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Transfected cells were lysed in ice-cold BN lysis buffer (1% (v/v) Triton X-100, 10% (v/v) glycerol, 20 mm bis-tris, 500 mm aminohexanoic acid, 20 mm NaCl, 2 mm EDTA, pH 7.0, 250 μl/well). The lysate was centrifuged at 21,000 × g at 4 °C for 10 min to remove insoluble material. The supernatant was collected and diluted 10-fold in Triton lysis buffer. 8% bis-tris-buffered polyacrylamide gels (0.75 mm) at pH 7.0 containing 66 mm aminohexanoic acid were used. Wells were topped up with cathode buffer (50 mm Tricine, 15 mm bis-tris, 0.02% (w/v) Coomassie G-250, pH 7.0). 10 μg of BSA was used as a molecular mass marker, giving bands at 66 and 132 kDa. Gels were run on ice at 100 V until samples entered the gel, then at 180 V until the Coomassie dye front reached the end of the gel. BN-PAGE gels were destained with 40% (v/v) methanol, 10% (v/v) glacial acetic acid for 30 min, refreshing the destaining solution every 10 min. Gels were soaked for 30 min in 1% SDS in Tris-buffered saline, pH 7.4, at room temperature. Proteins were blotted onto PVDF membrane by wet transfer at 100 V for 1 h. Coomassie-stained BSA marker bands were marked onto the membrane using a felt-tipped pen. Membranes were blocked in 20% (w/v) Marvel-skimmed milk powder in 0.1% PBS-Tween for 1 h. Membranes were incubated overnight at 4 °C on a roller in rabbit anti-AQP4 antibody (Abcam, ab128906) diluted 1:5,000 or rabbit anti-GFP (Abcam, ab6556) diluted 1:10,000, both in 5 ml of 0.1% PBS-Tween. Membranes were washed in 0.1% PBS-Tween and incubated with donkey anti-rabbit HRP (Santa Cruz, sc-2313) diluted 1:10,000 in 20 ml of 0.1% PBS-Tween at room temperature for 1 h. HRP was detected on x-ray film using ECL reagent (Amersham Biosciences). For multiple comparisons, one-way ANOVA was used, followed by post hoc t-tests with the p values subjected to Bonferroni correction for multiple comparisons. All data are presented as mean ± S.E. Simulations were done using the GROMOS 53A6 forcefield (14.Oostenbrink C. Villa A. Mark A.E. van Gunsteren W.F. A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6.J. Comput. Chem. 2004; 25: 1656-1676Crossref PubMed Scopus (2958) Google Scholar) extended to include lipid parameters (15.Berger O. Edholm O. Jähnig F. Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature.Biophys. J. 1997; 72: 2002-2013Abstract Full Text PDF PubMed Scopus (1602) Google Scholar) in Gromacs version 4.5.5 (16.Pronk S. Páll S. Schulz R. Larsson P. Bjelkmar P. Apostolov R. Shirts M.R. Smith J.C. Kasson P.M. van der Spoel D. Hess B. Lindahl E. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit.Bioinformatics. 2013; 29: 845-854Crossref PubMed Scopus (5101) Google Scholar). An AQP4 tetramer was generated according to the biological assembly entry in the AQP4 Protein Data Bank file 3GD8 (17.Ho J.D. Yeh R. Sandstrom A. Chorny I. Harries W.E. Robbins R.A. Miercke L.J. Stroud R.M. Crystal structure of human aquaporin 4 at 1.8 Å and its mechanism of conductance.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7437-7442Crossref PubMed Scopus (265) Google Scholar). N and C termini of the protein were truncated in the structure so proteins were simulated with neutral termini. The AQP4 tetramer was embedded into 5 pre-equilibrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers using inflateGRO (18.Schmidt T.H. Kandt C. LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations.J. Chem. Inf. Model. 2012; 52: 2657-2669Crossref PubMed Scopus (93) Google Scholar) and hydrated using Gromacs. Na+ and Cl− were added to a final concentration of 100 mm. Equilibration was achieved by steepest gradient energy minimization, 100-ps NPT simulation with 1,000 kJmol−1 nm−1 restraints on protein heavy atoms followed by three 1-ns NVT simulations with 1000, 100, and 10 kJmol−1 nm−1 restraints on protein heavy atoms followed by 30-ns unrestrained simulation. A Nosé-Hoover thermostat (0.5 ps, 310 K) was used to maintain constant temperature and 2 Parinello-Rahmann barostats (2 ps, 1 atm) were used to maintain constant pressure with zero surface tension. 1.4-nm cut-offs were applied for dispersion and short-range electrostatic interactions. Long-range electrostatics were treated using particle mesh Ewald. Hydrogen bonds were identified using the H-bonds plugin of visual molecular dynamics (19.Humphrey W. Dalke A. Schulten K. VMD: visual molecular dynamics.J. Mol. Graph. 1996; 14 (27–38): 33-38Crossref PubMed Scopus (38403) Google Scholar) using 3 Å and 20° cut-offs. Hydrogen bond occupancy was calculated according to these cut-offs at 100-ps intervals along the 100-ns trajectories and averaged over the 4 monomers and 5 trajectories. Plate reader-based calcein fluorescence quenching was done following Fenton et al. (20.Fenton R.A. Moeller H.B. Nielsen S. de Groot B.L. Rützler M. A plate reader-based method for cell water permeability measurement.Am. J. Physiol. Renal Physiol. 2010; 298: F224-F230Crossref PubMed Scopus (25) Google Scholar). MDCK cells were plated into black-walled, clear-bottomed tissue culture treated 96-well plates (Greiner) at 50% confluence 24 h before the experiment. Cells were loaded with 5 μm calcein-AM in growth medium supplemented with 1 mm probenecid (to inhibit dye leakage) for 90 min. Cells were washed twice with HEPES-buffered growth medium supplemented with 1 mm probenecid, then covered with 75 μl of probenecid-supplemented HEPES-buffered medium. Fluorescence was read on a BioTek synergy HT plate reader with injector system. Each well was read continuously (dt = 50 ms) for 5 s, followed by injection of 75 μl of HEPES-buffered medium containing 400 mm mannitol to give a final concentration of 200 mm and an osmotic gradient of 200 mosmol. Fluorescence was read for a further 50 s. Normalized fluorescence values were converted to normalized volumes using a Coulter counter generated standard curve. Single-phase exponential decay functions were fitted and rate constants were taken as proportional to the membrane water permeability. Using the crystal structure of AQP4 (17.Ho J.D. Yeh R. Sandstrom A. Chorny I. Harries W.E. Robbins R.A. Miercke L.J. Stroud R.M. Crystal structure of human aquaporin 4 at 1.8 Å and its mechanism of conductance.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7437-7442Crossref PubMed Scopus (265) Google Scholar), residues likely to form the tetrameric interface were identified, based on the physical distance between residues on adjacent monomers. Alanine substitution mutagenesis was used to investigate the contribution of these residues to oligomeric assembly. The identified residues were clustered into two regions. The first cluster comprises a patch of hydrophobic residues at the interfaces of TM1 and TM2 of one monomer with TM4 and TM5 of the adjacent monomer (Fig. 1A); the second cluster comprises 12 predominantly polar and charged residues forming intracellular loop D and the bottom of TM2 (Fig. 1B). 24 single point alanine-substitution mutants were made and six compound mutants were used to investigate the possibility of synergistic effects of several residues. There are two protein kinase A/C consensus sites in loop D (Ser180 and Ser188) so phosphomimetic mutations were also made (S180D and S188D). All mutants are listed in the left-hand column of Table 1. BN-PAGE followed by immunoblotting was used to assess the oligomeric state of all mutants expressed in HEK293 cells. Representative BN blots are shown in Fig. 2A and the effect of all mutations on oligomeric assembly and surface expression summarized in Table 1 and Fig. 2B. None of the hydrophobic residues in the hydrophobic patches had any effect on tetramer formation, either in isolation or in compound mutants. TM compound mutants consisted of simultaneous mutations of all residues identified within that transmembrane segment (e.g. TM1 denotes the simultaneous mutations I43A/I47A/L50A/L51A/I57A). Of the loop D single alanine mutants, only D179A had an effect on oligomeric assembly in isolation, and this effect was only minimal, with 95 ± 2% of the protein still assembled into tetramers. Unlike the hydrophobic cluster, the compound mutants of loop D had a clear effect on the ability of AQP4 to tetramerize. The two compound mutants, loop D1 (D179A/S180A/K181A/R182A/T183A) and loop D2 (D184A/V185A/T186A/G187A/S188A), caused reductions in oligomeric assembly: only 19 ± 4% of the loop D1 protein assembled into tetramers with both dimers (27 ± 5%) and monomers (53 ± 4%) being present, whereas the loop D2 protein predominantly formed dimers (67 ± 7%) with 33 ± 7% assembled into tetramers (n = 3).TABLE 1Oligomerization state and surface expression of AQP4 mutantsMutantOligomerization stateSurface expression% of WTI43AT113.9 ± 5.1I47AT95.8 ± 3.2L50AT102.5 ± 4.1L51AT96.1 ± 13.0I57AT104.1 ± 4.1TM1 (I43A/I47A/L50A/L51A/I57A)T86.9 ± 3.1L72AT121.1 ± 25.6L79AT88.9 ± 13.3TM2 (L72A/L79A)T97.7 ± 5.6L161AT91.9 ± 18.4I165AT87.9 ± 3.6TM4 (L161A/I165A)T85.4 ± 11.2I189AT97.7 ± 5.9I199AT96.2 ± 8.5F203AT112.4 ± 13.3TM5 (I189A/I199A/F203A)T93.3 ± 9.0Q86AT118 ± 4.4H90AT113.5 ± 4.1D179AT (95 ± 2%)87.6 ± 19.4D (5 ± 2%)S180AT109.8 ± 4.5S180DT99.7 ± 4.9K181AT103.2 ± 4.5R182AT96.1 ± 22.1T183T105.6 ± 3.6Loop D1 (D179A/S180A/K181A/R182A/T183A)T (19 ± 4%)88.5 ± 6.1D (27 ± 5%)M (53 ± 4%)D184AT97.7 ± 8.8V185AT102.2 ± 5.4T186AT95.1 ± 7.5G187AT106.7 ± 5.1S188AT98.6 ± 3.2S188DT104.1 ± 4.2Loop D2 (D184A/V185A/T186A/G187A/S188A)T (33 ± 7%)83.1 ± 11.0D (67 ± 7%) Open table in a new tab FIGURE 2.BN-PAGE and Western blotting of AQP4 mutants. A, representative Western blots following BN-PAGE of Triton X-100-solubilized AQP4 mutants, showing the effect of the loop D1 and loop D2 compound mutations, and a lack of effect of mutations on the transmembrane hydrophobic patch. 66 and 132 denote the positions of BSA molecular weight marker bands. The AQP4-GFP construct, including linker peptide, has a predicted molecular mass of 63.1 kDa. B, percentage of protein assembled into tetramers calculated using densitometry following BN-PAGE and Western blotting. Effective mutations are highlighted with white bars. Data are presented as mean ± S.E. from 3 experimental repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There was no significant difference in the surface expression of the loop D mutants compared with wild-type AQP4. Surface expression was assessed qualitatively by live cell confocal microscopy using GFP-tagged AQP4 mutant constructs and quantitatively by cell surface biotinylation. Fig. 3A shows representative confocal micrographs of HEK293 cells transfected with GFP fusion proteins of AQP4 wild-type, and the loop D1 and loop D2 mutants. Surface expression in transiently transfected HEK293 cells measured by cell surface biotinylation was not significantly different (p = 0.53, one-way ANOVA, n = 3) for either of the loop D compound mutants (D1 and D2) or the single alanine mutants (Fig. 3B). This suggests that the trafficking machinery is able to interact with non-tetrameric aquaporins. The cell surface biotinylation data are summarized for all mutants in the third column of Table 1. It was important to confirm the reduction in tetramerization of AQP4 molecules that had been constitutively trafficked to the cell surface and to rule out intracellular retention of dimeric/monomeric species or changes in detergent sensitivity caused by loop D substitutions. Several complementary biophysical techniques were used to address this. Recovery curves were collected from 5 different cells per experimental repeat (n = 6). Representative fluorescence recovery curves are shown in Fig. 3D, along with the average recovery half-times. From these, diffusion coefficients were calculated: 5.2 ± 0.3 × 10−3 μm2 s−1 (AQP4 WT), 5.9 ± 0.3 × 10−3 μm2 s−1 (loop D1), and 5.8 ± 0.2 × 10−3 μm2 s−1 (loop D2). Both loop D mutant diffusion coefficients were significantly different from the wild-type (D1 p = 0.01 and D2 p = 0.02 by Student's t test following ANOVA, with p values subjected to Bonferroni correction). The FRAP data suggest reduced tetramerization for the surface-localized loop D mutants, although the increased diffusion coefficient could also be explained by the inability of these mutants to form a complex with a third party protein. To complement these FRAP experiments, biotinylated cell surface proteins were isolated using neutravidin-coated plates, eluted by reducing the S-S bond incorporated into the biotinylation reagent (using 1% β-mercaptoethanol in BN lysis buffer) and subjected to BN-PAGE (representative blots are shown in Fig. 3C). Surface-localized mutant AQP4 molecules subjected to BN-PAGE had the same changes in tetramerization seen in whole cell lysates (n = 3). To complement the above analyses, AQP4 constructs tagged with Venus (a yellow fluorescent protein (YFP) derivative) and mTurquoise2 (a cyan fluorescent protein (CFP) derivative) were generated and co-transfected into HEK293 cells to form a FRET biosensor for homo-oligomerization in living cells. The wild-type AQP4 constructs gave a robust FRET signal with an average apparent efficiency of 44.2 ± 3.6% (Fig. 4). We were unable to measure any FRET in cells co-transfected with AQP1-Venus and AQP4-Turquoise despite high co-localization (data not shown), suggesting that the FRET interactions occur primarily within the AQP4 tetramers and not between tetramers that are transiently close together in the plane of the membrane. The probability of a particular donor molecule taking part in FRET is dependent on the number of acceptors within the Forster radius and vice versa. For CFP-YFP, the Forster radius is ∼5 nm (21.Felber L.M. Cloutier S.M. Kündig C. Kishi T. Brossard V. Jichlinski P. Leisinger H.J. Deperthes D. Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations.BioTechniques. 2004; 36: 878-885Crossref PubMed Google Scholar). Based on the AQP4 crystal structure, the monomer-monomer center of mass separations are 2.8 (adjacent monomers) and 3.9 nm (diagonal monomers), respectively, so both would be expected to contribute to the FRET signal (assuming that the average separation of the C-terminal tails is similar). The average number of FRET pairings in a sample of co-transfected cells is therefore dependent on the level of AQP4 oligomerization. Both D1 and D2 compound mutants had a slightly larger than 2-fold reduction in FRET efficiency (to 17 ± 6 and 20 ± 4%, respectively, p = 0.003 and p = 0.005, n = 4) compared with the wild-type (Fig. 5A), suggesting that these constructs have a reduced propensity to oligomerize in live cells, further confirming that the changes seen in the BN-PAGE were not mediated by changes in detergent sensitivity. Furthermore, for the mutants, there was no difference in FRET efficiency between plasma membrane and intracellular membranes (Figs. 5, B and C), suggesting that the oligomerization state of these m" @default.
• W2283938713 created "2016-06-24" @default.
• W2283938713 creator A5009542352 @default.
• W2283938713 creator A5020224635 @default.
• W2283938713 creator A5056447859 @default.
• W2283938713 creator A5085722901 @default.
• W2283938713 date "2016-03-01" @default.
• W2283938713 modified "2023-10-17" @default.
• W2283938713 title "Structural Determinants of Oligomerization of the Aquaporin-4 Channel" @default.
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