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 a
Trang 1sequence signature motifs for cation exclusion
Dorothea Wree1, Binghua Wu1, Thomas Zeuthen2and Eric Beitz1
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 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.
Trang 2experimental 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 Asnfi 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 BurkholdeBurkholde-ria 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.
Trang 3The 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-cepaciaaquaglyceroporin [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 selectivpermeabil-ity, 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-siaeyeast 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.
Trang 4Glycerol 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 kcalÆmol)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 (pKa= 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 (Pf) and glycerol (Pgly) For evaluation, six
to 10 traces from each of two independent experiments were
aver-aged The error bars denote standard error of the mean.
Trang 5determined 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 Asnfi 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.
Trang 6Permeability 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 Asnfi 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 Ncap position: Asn, Asp, Ser,
and Thr The following Ncap+1 position is typically
occupied by a Pro, whereas the degree of variation at
Ncap+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 Ncap
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 Ncap 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 Ncap 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 Ncap 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 Ncap 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
Trang 7Collection 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
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
and the membranes were collected from the supernatants
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
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
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 Pf was calculated from Pf= 1⁄ sV0⁄ (S0VWCdiff)
[31], where s is the time constant of the exponential fitting
the partial molar water volume (18 cm3Æmol)1), and Cdiffthe concentration of the osmotically active solute after mixing
measured by mixing with glycerol buffer (incubation buffer supplemented with 0.6 m glycerol) The glycerol permeability
light scattering curve, using Pgly= |dI⁄ dt|(V0Cout)⁄ (S0Cdiff), where dI⁄ dt is the slope of the intensity curve, V0and S0are
(1.5 m), and Cdiffis the chemical glycerol gradient (0.3 m) In each experiment, six to 10 trace curves were recorded and
for calculation of the Arrhenius activation energy
Phenotypic S cerevisiae methylamine efflux assay
The assay was performed as described previously [2] In
BccGlpF and mutants, or EcGlpF, were grown overnight
cultures were harvested by centrifugation (16 000 g, 30 s),
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
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,
10 mm Hepes or Mes, pH 7.4) or with test solutions at a
Trang 8rate of 20 mLÆmin)1 To measure the H+permeability, the
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 Beitz) and
grants from the Danish Research Council, the
Lund-beck Foundation, and Loevens (to T Zeuthen)
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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
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