Báo cáo khoa học: Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion docx

Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion Dorothea Wree 1 , Binghua Wu 1 , Thomas Zeuthen 2 and Eric Beitz 1 1 Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Germany 2 Institute of Cellular and Molecular Medicine, University of Copenhagen, Denmark Introduction Aquaporins (AQPs) of the orthodox, water-specific subfamily and of the water-permeable and solute-per- meable aquaglyceroporin subfamily share a common protein fold [1]. It comprises six membrane-spanning helices plus two half-helices with their positive, N-ter- minal ends located at the centre of the protein and their C-terminal ends pointing towards either side of the membrane. The helices surround the 20-A ˚ -long and 3–4-A ˚ -wide amphipathic AQP channel. Two con- served constriction sites are present in the channel. The aromatic ⁄ Arg (ar ⁄ R) constriction is located at the extracellular pore mouth. Its diameter determines whether or not solutes, such as glycerol and methyl- amine, can pass the AQP in addition to water [2–5]. Furthermore, the positively charged residues in this region form an energy barrier for protons [2,3,5]. The role in pore selectivity of the ar ⁄ R constriction is well understood, owing to several theoretical and Keywords aquaglyceroporin; aquaporin; potassium; proton; sodium Correspondence E. Beitz, Pharmaceutical Institute, Christian- Albrechts-University of Kiel, Gutenbergstrasse 76, 24118 Kiel, Germany Fax: +49 431 880 1352 Tel: +49 431 880 1809 E-mail: ebeitz@pharmazie.uni-kiel.de Website: http://www.pharmazie.uni-kiel.de/ chem/ (Received 8 November 2010, revised 10 December 2010, accepted 13 December 2010) doi:10.1111/j.1742-4658.2010.07993.x Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic ⁄ Arg (ar ⁄ R) con- striction, has been well characterized by mutational analysis, experimental data concerning the NPA region – in particular, the Asn position – is miss- ing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeabil- ity. However, other mutations in the NPA region led to reduced permeabil- ity (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability. Abbreviations AQP, aquaporin; ar ⁄ R, aromatic ⁄ Arg; BccGlpF, Burkholderia cenocepacia glycerol facilitator; Ch, choline; EcGlpF, Escherichia coli glycerol facilitator; HA, haemagglutinin. 740 FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS experimental studies, which have elucidated contribu- tions of the individual residues at this site. The second constriction resides in the centre of the channel, where the positive ends of the two half-helices meet. The helix dipole moments add up to a full positive charge, and the resulting electrostatic field poses another energy barrier for cations [6]. The residues that constitute the capping structures of the half-helices are extremely well conserved in the two canonical NPA motifs. Although there is some degree of variation in the second and third positions [7–9], the Asn at the first position appears to be almost invariable [10,11]. The Asn side chain amide moieties fulfil two roles: (a) the carbonyl oxygens form hydrogen bonds with backbone nitrogen atoms of the preceding two amino acids, stabilizing the helix cap; and (b) the amide nitrogens act as hydrogen bond donors to passing water or solute molecules, and may thus be involved in AQP selectivity. We identified, in the genomes of Burkholderia sp. [12], genes encoding aquaglyceroporins that intrinsi- cally have SPA at the second NPA motif position. The natural occurrence of an Asn fi Ser exchange led us to analyse the functional consequences of Asn replace- ments at the NPA sites by site-directed mutagenesis. As expected, we found that the Asn positions are structurally critical. However, the Asn positions in both NPA motifs can be occupied by Ser, yielding functional AQPs with unaltered water and solute permeability. However, Ser leads to a sodium leak in mammalian AQP1, which may explain why 99% of all AQPs carry Asn at the NPA sites. Results Natural replacement of Asn in the NPA motifs is rare Inspection of the b-proteobacterial genome data from Burkholderia species, i.e. Burkholderia cenocepacia [12], Burkholderia cepacia, Burkholderia mallei, Burkholde- ria pseudomallei, and Burkholderia fungorum, yielded a family of putative aquaglyceroporin genes encoding proteins with unusual NPA motifs (Fig. S1). The sec- ond NPA motif appeared to be altered to SPA, whereas the remaining sequences were 38% identical and 58% similar to the prototypical aquaglyceroporin from Escherichia coli [E. coli glycerol facilitator (EcGlpF)] [13]. We then analysed 1357 AQP sequences deposited in the RefSeq database [14], and identified only 15 (1.1%) with a substitution of one of the Asn residues in the NPA motifs by Ser or Cys, which is in line with the findings of an earlier study [11]. A fre- quency-corrected sequence logo [15] shows the strong conservation of the NPA motifs and of the direct sequence vicinity (Fig. 1A, top). To search for addi- tional characteristic amino acid exchanges in the Burk- holderia aquaglyceroporin subfamily, we generated a subfamily logo [16], which displays sequence deviations at alignment positions with high information content, i.e. at conserved positions. The result for the NPA regions is shown in Fig. 1A (lower panel). Residues of the subfamily are displayed upright, whereas residues of the remaining set of proteins appear upside-down. A B Fig. 1. Sequence comparison and structure model of the NPA ⁄ SPA region of BccGlpF. (A) The upper panel depicts a sequence logo of the AQP family, showing conservation of the five residues upstream and downstream of the Asn position of either NPA motif (labelled with black bars). The subfamily logo [16] below indicates residues that are characteristic for the Burkholderia aquaglyceropo- rin subfamily (upright symbols), and the upside-down letters indi- cate the corresponding residues of the remaining set of AQPs. The more distinct a residue, the more information is contained at this site, as reflected by the height of the symbol. The actual BccGlpF sequence is given below the logos. (B) Structural model of the BccGlpF NPI-SPAR sequence region based on EcGlpF (Protein Data Bank: 1FX8) [13]. Asn64, Ser186 Arg189 and the indicated carbonyl oxygens of the protein backbone represent the hydrophilic inter- action sites along one side of the otherwise hydrophobic AQP channel. NPI; SPAR. D. Wree et al. Role of asparagine in the aquaporin NPA motif FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS 741 The height of a residue symbol reflects the subfamily’s degree of distinction at this site. Ser186 turned out to be the most characteristic residue for the Burkholderia aquaglyceroporins (4.9 bit). Other prominent positions, such as Asp190 (2.7 bit), denoted residues that are gen- eral discriminators between orthodox AQPs and aqua- glyceroporins [17]. The exchange of Asn for Ser in the NPA region may have structural and functional consequences. Hence, we generated a structure model of the B. ceno- cepacia aquaglyceroporin [B. cenocepacia glycerol facil- itator (BccGlpF)], by mapping the protein sequence on the 2.2-A ˚ resolution crystal structure of EcGlpF (Pro- tein Data Bank: 1FX8) [13] (Fig. 1B). Being shorter by one methylene group, Ser186 enlarges the diameter in the NPA region by about 20% of the average diameter of the remaining channel, leaving the ar⁄ R region around Arg189 as the only constriction in the channel path. Ser186 may form two stabilizing hydrogen bonds between its hydroxyl oxygen and the backbone amide nitrogens at the preceding two amino acid positions, similar to the hydrogen bonds between the Asn64 car- bonyl oxygen and the backbone of the second half- helix (green dotted lines in Fig. 1B). The Asn64 side chain amide also provides two hydrogen donor sites for interaction with passing water and solute molecules, whereas the Ser186 hydroxyl moiety acts as a donor for a single hydrogen bond. It is not clear whether this hydrogen is accessible from within the channel, owing to major differences in its position and orientation as compared with the hydrogens of an Asn side chain amide. To address the question of whether the presence of an SPA motif in BccGlpF affects channel permeabil- ity or selectivity, we cloned the respective ORF from genomic DNA of B. cenocepacia for site-directed muta- genesis, expression, and functional analysis. Expression of wild-type BccGlpF and mutants Like to other bacterial AQPs, BccGlpF was not expressed in Xenopus laevis oocytes. However, we obtained good expression in the Saccharomyces cerevi- siae yeast system (Fig. 2), which was used for the fol- lowing functional analysis. We generated several BccGlpF mutants in which Asn64 was changed to Ala, Asp, or Ser, and Ser186 was changed to Ala, Asn, Asp, Cys, or Thr, and one double mutant with switched positions of Asn and Ser, i.e. N64S ⁄ S186N. We chose as substitutes only small residues with side chains smaller than or the same size as the Asn side chain, because it has been shown in an early AQP study that slightly larger residues, such as Gln, impair channel function [18], and a major change to Lys was found to suppress expression of AQP1 in humans, leading to a Colton-null phenotype [19]. Comparison of the BccGlpF mutant expression levels in yeast by semiquantitative densitometry of western blots showed three categories (Fig. 2): (a) expression level similar to that of wild-type BccGlpF (N64A, N64S, N64S ⁄ S186N, and S186N); (b) five-fold to 10-fold reduced expression (N64D, S186C, and S186D); and (c) undetectable expression (S186A and S186T). Dimers of 54 kDa and higher-order complexes of the expressed AQPs were visible when sufficient protein was loaded. Water and glycerol permeability of wild-type BccGlpF and mutants To test for water permeability, we expressed wild-type BccGlpF and mutants in a yeast strain that lacked the endogenous aquaglyceroporin S. cerevisiae glycerol facilitator [20], prepared yeast protoplasts, and sub- jected them to an outward-directed osmotic sorbitol gradient of 300 mosmÆkg )1 in a rapid mixing device. The resulting cell shrinkage was determined by moni- toring the relative increase in light scattering (Fig. 3A, left panel). Here, only the control cells expressing mammalian AQP1 [21] showed a rapid cell volume change caused by water efflux, which was 15-fold fas- ter than that of nonexpressing cells (Fig. 3A, left panel and insert). Expression of wild-type BccGlpF or mutants did not increase the water flux above that of cells without AQP expression (Fig. 3B, left panel). Similarly, the water permeability of EcGlpF was too low to be detected, which is consistent with earlier studies that have shown a one order of magnitude lower water permeability of EcGlpF than that of water-specific AQPs [22]. Fig. 2. Relative expression levels of wild-type (wt) BccGlpF and mutants in yeast by western blot. The proteins were detected via N-terminal HA-tags and a specific antiserum. The bands at 54 kDa correspond to protein dimers. Higher-order complexes can also be seen. Role of asparagine in the aquaporin NPA motif D. Wree et al. 742 FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS Glycerol permeability was measured with the same cells and an outward-directed osmotic glycerol gradient of 300 mosmÆkg )1 . Under these conditions, the protop- lasts first shrunk because of water efflux. Subsequently, in a second phase, the volume recovered partially in the presence of functional glycerol channels, owing to glycerol influx following the chemical gradient (Fig. 3A, right panel). We chose this biphasic setup because an isotonic glycerol gradient as typically used with Xenopus oocytes [7] did not yield a robust assay system. EcGlpF served as a positive control, showing the expected volume recovery effect (Fig. 3A, right panel and insert). BccGlpF exhibited the same degree of glycerol permeability as EcGlpF (Fig. 3B, right panel), confirming functionality of the novel Burk- holderia aquaglyceroporin. Analysis of the BccGlpF mutants with any combination of Asn and Ser in both NPA motifs (N64S, S186N, and N64S ⁄ S186N) showed equal glycerol permeability as obtained with the wild type, and Arrhenius activation energies of approxi- mately 6 kcalÆ mol )1 , whereas the remaining mutants (N64A, N64D, S186C, S186D, and S186T) were non- functional (Fig. 3B, right panel). Together, BccGlpF water and glycerol permeability are comparable to those seen with GlpF, and Asn and Ser are interchangeable in both BccGlpF NPA motifs without affecting glycerol permeability. Methylamine permeability of wild-type BccGlpF and mutants To test for solute selectivity of the BccGlpF mutants, we employed a sensitive phenotypic yeast assay for methylamine permeability [2]. Methylamine is an ana- logue of ammonia and, similarly, its protonation status depends on the environmental pH (pK a = 10.6). For example, at pH 6.5, only 0.008% of the compound will be in the unprotonated methylamine form, whereas 99.992% will be protonated as methylammonium. Yeast cells endogenously express ammonium transport- ers of the S. cerevisiae methylamine permease family, which transport protonated methylammonium into the cells independently of the external pH [23]. As methy- lammonium is toxic to the yeast, the cells can only sur- vive when the compound is immediately shuttled out again. Aquaglyceroporins have been shown to pass unprotonated methylamine if a pH gradient is gener- ated from an intracellular pH 6.8 to a more acidic external pH. Accordingly, yeast expressing EcGlpF grows well on methylammonium-containing agar plates at pH 5.5, whereas a flat pH gradient (pH 6.5) allows for only weak growth (Fig. 4). Cells without an aqua- glyceroporin do not grow, owing to accumulation of toxic methylammonium. Wild-type BccGlpF and the same set of mutants that conducted glycerol (N64S, S186N, and N64S ⁄ S186N) rescued yeast growth, con- firming functionality of these channels (Fig. 4). How- ever, we found that three more mutants that were impermeable for glycerol conducted the smaller methylamine, i.e. BccGlpF N64A, S186C, and S186D. Cell growth of yeast expressing the BccGlpF N64A mutant was as high as that of yeast with wild-type BccGlpF, whereas yeast expressing the BccGlpF S186C or S186D mutants grew considerably more slowly (Fig. 4), correlating with the expression levels A B Fig. 3. Water and glycerol permeability of wild-type (wt) BccGlpF and mutants. (A) Changes in light scattering of yeast protoplasts in a 300 m M osmotic sorbitol gradient for measuring water permeabil- ity (left panel) or in a 300 m M osmotic glycerol gradient for glycerol permeability (right panel). Nonexpressing cells (–) and cells express- ing rat AQP1 (for water) or EcGlpf (for glycerol) were used as con- trols. The parts of the traces that are relevant for calculation of the permeability coefficients are enlarged in the inserts. (B) Permeabil- ity coefficients for water (P f ) and glycerol (P gly ). For evaluation, six to 10 traces from each of two independent experiments were aver- aged. The error bars denote standard error of the mean. D. Wree et al. Role of asparagine in the aquaporin NPA motif FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS 743 determined by western blot (Fig. 2). Again, the remaining BccGlpF mutants (N64D, S186A, and S186T) were nonfunctional. Water and ion permeability of a mammalian AQP1 N76S mutant Having established that Asn fi Ser exchanges in the NPA motifs of BccGlpF do not alter glycerol and methylamine permeability, we investigated whether water permeability or ion exclusion might be affected. BccGlpF, however, could not be studied effectively in the respective assays: the water permeability was too low, and the protein was not made in Xenopus oocytes. As an alternative, we generated an analogous AQP1 N76S mutant that is well expressed in Xenopus oocytes. Also, ion permeability of AQPs has been found and described only in selected mutants of AQP1, such as AQP1 R195V [2,5]. The AQP1 R195V mutant mimicks the situation found in the group of AQPs that carry only uncharged residues in the ar ⁄ R pore constriction. Together, the AQP1 N76S single mutant and AQP1 N76S ⁄ R195V double mutant allowed us to study the effect of an SPA motif on water, proton, potassium and sodium permeability, and to compare the results with previously established data [2] (Fig. 5). The water permeability of the AQP1 N76S mutant was identical to that of wild-type AQP1 and the AQP1 R195V mutant (Fig. 5, upper left panel) and 20-fold higher than that obtained with nonexpressing control oocytes. The AQP1 N76S ⁄ R195V double mutant showed a 32% reduction in water permeability, which is similar in trend to a former AQP1 N76D ⁄ H180A ⁄ R195V mutant, which exhibited an 86% reduction [5]. We then measured the ion conductance of wild-type AQP1 and the mutants in comparison with nonex- pressing control oocytes, using two-electrode voltage- clamp and a protocol described previously [2,5]. For testing of proton conductance, an inward gradient was established by shifting the bath pH to 5.5. However, the currents obtained were not significantly different in control oocytes (not shown), wild-type AQP1-express- ing oocytes, and AQP1 N76S-expressing oocytes (Fig. 5, upper right panel). This indicates that the AQP1 N76S mutant is impermeable for protons. How- ever, we reproduced the proton leak of the AQP1 R195V mutant [2], which was further enhanced in the AQP1 N76S ⁄ R195V double mutant by a factor of 3. Fig. 4. Phenotypic yeast assay for methylamine permeability of wild-type BccGlpF and mutants. Cell growth at acidic pH indicates efflux of toxic methylamine from the cells via the expressed aqua- glyceroporins. Nonexpressing cells (–) and cells expressing EcGlpF were used as controls. The control plate without addition of methyl- amine demonstrates even loading of the samples. Fig. 5. Water, proton, potassium and sodium permeability of wild- type (wt) AQP1, and AQP1 ar ⁄ R or NPA mutants, in X. laevis oocytes. Water permeability (upper left panel) was calculated from oocyte shrinkage in medium supplemented with 20 m M mannitol. Control oocytes without AQP1 expression (native) showed 20-fold lower water permeability. For cation permeability, a two-electrode voltage clamp setup was used, with a voltage stepping protocol from +40 mV to )120 mV, and a 150-ms duration of each step. The steady-state currents were recorded after 100 ms. Respective cation gradients were generated by a pH shift in the bath from 7.4 to 5.5 (upper right panel; for proton permeability), replacement of 25 m M Ch in the bath by potassium (lower left panel), or replace- ment of 50 or 100 m M Ch for sodium (lower right panel). Cation permeability of control oocytes without AQP1 expression was not significantly different from that of oocytes expressing wild-type AQP1, and are not shown. Error bars denote standard errors of the mean. The asterisks indicate values that are significantly different from those obtained with wild-type AQP1. Role of asparagine in the aquaporin NPA motif D. Wree et al. 744 FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS Permeability for alkali cations was measured by par- tial, isotonic replacement of impermeable choline (Ch) in the bathing solution with potassium or sodium, and application of the voltage stepping protocol. Significant potassium currents above those of control oocytes were not detectable in any of the tested AQP1 variants. How- ever, expression of the AQP1 N76S mutant robustly increased the sodium current two-fold over control or AQP1-expressing oocytes, and we even observed a five- fold increase with the AQP1 N76S ⁄ R195V mutant. The AQP1 R195V mutant was impermeable for sodium ions. In summary, our data show that Asn fi Ser exchanges in the NPA motifs are well tolerated during protein biosynthesis, and that the resulting AQP chan- nels display normal water and solute permeability but leak sodium ions. Discussion Various statistical analyses of data from protein struc- ture databases have ranked the 20 proteinogenic amino acids according to their frequency at N-terminal helix caps [24–27]. Accordingly, mainly four residues are strongly preferred at the N cap position: Asn, Asp, Ser, and Thr. The following N cap+1 position is typically occupied by a Pro, whereas the degree of variation at N cap+2 increases drastically. The findings are in strik- ing agreement with the situation found at the N-termi- nal ends of the characteristic AQP half-helices, which carry canonical NPA motifs with an almost invariable Asn position and somewhat less conserved Pro and Ala positions [7–11]. Averaged over the full set of proteins in the database, a helix cap position does not display a preference among Asn, Asp, Ser, or Thr. However, in the subset of AQP half-helices, 99% of the N cap posi- tions are filled with an Asn, and the remaining 1% is shared between Ser and probably Cys, with the Cys being predicted but not yet experimentally confirmed. What is the reason for this strong preference for Asn? Considering the spatial restrictions in the centre of the AQP channel, it seems evident that only residues smaller than or of the same size as Asn are tolerated in the half-helix N cap position. Indeed, larger residues at these sites have been shown to block the AQP1 channel or even to massively interfere with AQP1 expression [18,19]. Despite its small size, Thr appeared to fully abolish expression of the respective AQP mutant. This phenomenon may be explained by the b-branched molecular structure of Thr, which can interfere with protein function [28,29]. The branching next to the car- bon atom carrying the amino group may clash with the dense packing in the NPA protein region. Putting an Ala at the N cap position produced ambiguous results. Replacement of Asn by Ala at the first NPA site was fully compatible with protein expression, and even pro- duced to a functional channel with methylamine perme- ability. However, the permeability profile was altered because glycerol was no longer conducted. This may hint at influences on the channel structure, probably owing to the lack of stabilizing hydrogen bonds between the N cap residue – Ala is neither donor nor acceptor – and the half-helix backbone. Higher flexibil- ity in this region may prevent the larger glycerol from passing, whereas methylamine is still compatible with the slightly changed situation. The effect of S186C and S186D may be similarly explained. Ala at the N cap posi- tion of the second NPA motif (SPA in BccGlpF) was not tolerated, yielding no protein. Hence, the second NPA site appears to be structurally more critical than the first NPA site. This may be related to the immedi- ately following Arg as a major constituent of the criti- cal selectivity filter at the ar ⁄ R constriction (Fig. 1B). In contrast to replacement by Ala and Thr, replace- ment of Asn by Ser in either NPA motif produced a fully functional AQP. However, Ser is a rare residue at the AQP half-helix caps. Our finding of a sodium leak in the AQP1 N76S mutant provides an argument for the strong preference for Asn. Steady sodium leak cur- rents across the cell membrane require active export of sodium from the cytosol by ATPases, in order to maintain the cell’s resting potential, and thus interfere with bioenergetics [30]. Even a small additional leak enhances the energetic costs of the cell, and therefore represents an evolutionary disadvantage. We have shown previously that replacement of Asn in the NPA motifs by Asp generates a sodium leak, which is four- fold larger than that with Ser [5]. Asp and Ser carry oxygen atoms in their side chains as putative coordina- tion sites for sodium ions, and so may interfere with the electrostatic barrier function of the half-helix dipoles by increasing the probability of the presence of a sodium ion in the channel centre. It is tempting to analyse natural AQPs with predicted DPA as well as CPY motifs [11] with regard to cation exclusion. Together, Asn residues appear to be optimal in the N-capping NPA motifs of the AQP half-helices with regard to protein stability and cation exclusion, but not in terms of solute selectivity. Experimental procedures Cloning and site-directed mutagenesis of BccGlpF The ORF of the BccGlpF-encoding gene was amplified by PCR from genomic B. cenocepacia DNA (German D. Wree et al. Role of asparagine in the aquaporin NPA motif FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS 745 Collection of Microorganisms and Cell Cultures, DSMZ) and cloned into the yeast expression plasmid pDR196. An N-terminal haemagglutinin (HA) epitope tag was inserted via a synthetic SpeI–HA–PstI oligonucleotide dimer into respective sites of the plasmid. Point mutations were intro- duced with the QuikChange protocol (Stratagene, Heidel- berg, Germany) and primers with respective nucleotide exchanges. Correct amplification and mutagenesis were confirmed by DNA sequencing. A primer list is available from the authors upon request. Expression of BccGlpF in S. cerevisiae and membrane preparation for western blot BY4742Dfps1 (MATa his3D1 leu2D0 ura3D0 fps1::Kan- MX4) yeast cells (Euroscarf, Frankfurt, Germany) were transformed with the generated pDR196–BccGlpF con- structs. Single colonies were picked and grown overnight in liquid SD medium (–Ura) to a D 600 nm of 1–2. The cultures were harvested by centrifugation (4 °C, 2200 g, 5 min), washed with ice-cold water and extraction buffer (5 mm EDTA, 25 mm Tris, pH 7.5) plus protease inhibitor cock- tail (Roche, Mannheim, Germany), resuspended in extrac- tion buffer, and vortexed with acid-washed glass beads. The lysates were cleared by centrifugation (4 ° C, 1000 g, 5 min), and the membranes were collected from the supernatants (4 °C, 100 000 g, 45 min). Protein concentrations were determined by use of the Bradford method, with BSA as a standard. For semiquantitative densitometric analysis of the expression levels in yeast, equal amounts of total protein were separated by SDS ⁄ PAGE, checked for even loading of the lanes by Coomassie Blue staining, blotted onto poly(vinylidene difluoride) membranes, and detected with a monoclonal mouse antibody against the N-terminal HA epitope tags (Roche). Measurement of water and glycerol permeability by stopped-flow assay Yeast protoplasts were prepared from cells expressing wild- type BccGlpF, BccGlpF mutants, EcGlpF or AQP1 by digesting the cell wall with zymolyase-20T according to Bertl et al. [31], and stored in incubation buffer (50 mm NaCl, 5mm CaCl 2 , 1.2 m sorbitol, 10 mm Tris, pH 7). For mea- surement of water permeability, the suspension was rapidly mixed with an equal volume of osmotic buffer (incubation buffer supplemented with 0.6 m sorbitol) in a stopped-flow apparatus (SFM-300; BioLogic, Claix, France). Cell volume changes were monitored by measuring the intensity of 90° light scattering at 546 nm. The osmotic water permeability coefficient P f was calculated from P f =1⁄ sV 0 ⁄ (S 0 V W C diff ) [31], where s is the time constant of the exponential fitting function, V 0 the initial mean protoplast volume (65.45 lm 3 ), S 0 the initial mean protoplast surface area (78.54 lm 2 ), V W the partial molar water volume (18 cm 3 Æmol )1 ), and C diff the concentration of the osmotically active solute after mixing (300 mm or 3 · 10 )4 molÆcm )3 ). Glycerol permeability was measured by mixing with glycerol buffer (incubation buffer supplemented with 0.6 m glycerol). The glycerol permeability coefficient (P gly ) was calculated from the second phase of the light scattering curve, using P gly =|dI ⁄ dt|(V 0 C out ) ⁄ (S 0 C diff ), where dI ⁄ dt is the slope of the intensity curve, V 0 and S 0 are as above, C out is the total external solute concentration (1.5 m), and C diff is the chemical glycerol gradient (0.3 m). In each experiment, six to 10 trace curves were recorded and averaged. Measurements were performed at 20 °C and 36 ° C for calculation of the Arrhenius activation energy. Phenotypic S. cerevisiae methylamine efflux assay The assay was performed as described previously [2]. In brief, BY4742Dfps1 yeast cells expressing wild-type BccGlpF and mutants, or EcGlpF, were grown overnight at 29 °C in SD medium (– Ura) to a D 600 nm of 1–2. The cultures were harvested by centrifugation (16 000 g, 30 s), adjusted to a D 600 nm of 1 in water, and spotted in serial 1 : 10 dilutions on SD agar medium supplemented with 3% glucose, 0.1% proline as the sole nitrogen source, and 50 mm methylamine at pH 5.5 and 6.5. Cell growth was monitored after 5 days. Expression of rat wild-type AQP1 and mutants in X. laevis oocytes and water permeability Rat AQP1 and the AQP1 R195V mutant in the pOG1 vec- tor have been described previously [2]. The N76S point mutation was introduced with the QuikChange protocol (Stratagene). cRNA synthesis was performed with NotI lin- earized pOG1 plasmid with the mMessage mMachine T7 kit (Ambion, Darmstadt, Germany). Five nanograms of cRNA in 50 nL of water was injected into collagenase A (Roche)-defolliculated stage V and VI X. laevis oocytes. The oocytes were incubated for 3 days at 15 °C in ND96 buffer (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl 2 ,1mm MgCl 2 ,5mm Hepes, pH 7.4). Control oocytes were water- injected. Osmotic water permeability (L P ) was determined within 10 s after addition of 20 mm mannitol to the bathing solution from the rate of oocyte shrinkage [2]. Electrophysiology To measure cation-induced currents in Xenopus oocytes, we used the two-electrode voltage-clamp technique as described previously [2,5]. In short, microelectrodes were inserted into oocytes superfused with control solution (100 mm ChCl, 20 mm mannitol, 2 mm KCl, 1 mm CaCl 2 ,1mm MgCl 2 , 10 mm Hepes or Mes, pH 7.4) or with test solutions at a Role of asparagine in the aquaporin NPA motif D. Wree et al. 746 FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS rate of 20 mLÆmin )1 . To measure the H + permeability, the pH of the bathing solution was changed to 5.5; for Na + per- meability, 50 mm or 100 mm Ch + was replaced by Na + ; for K + permeability, 25 mm Ch + was replaced by K + . Before and after the solution change, the voltage of the voltage- clamped oocyte was jumped to potentials between +40 and )140 mV in steps of 20 mV lasting 150 ms. Corresponding steady-state clamp currents were recorded after 100 ms. Acknowledgements We thank B. Henke and C. Steinbronn for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft Be2253 ⁄ 3 (to E. 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Supporting information The following supplementary material is available: Fig. S1. Alignment of Burkholderia aquaglyceroporins in comparison with Escherichia coli glycerol facilitator (EcGlpF). This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Role of asparagine in the aquaporin NPA motif D. Wree et al. 748 FEBS Journal 278 (2011) 740–748 ª 2011 The Authors Journal compilation ª 2011 FEBS . Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion Dorothea Wree 1 , Binghua Wu 1 , Thomas Zeuthen 2 and. [28,29]. The branching next to the car- bon atom carrying the amino group may clash with the dense packing in the NPA protein region. Putting an Ala at the