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:
Website: />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. 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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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