COMMUNICATION
Switch from an Aquaporin to a Glycerol Channel by Two Amino Acids
Substitution*
Valérie
Lagrée,
Alexandrine
Froger,
Stéphane
Deschamps,
Jean-François
Hubert,
Christian
Delamarche,
Georgette
Bonnec,
Daniel
Thomas,
Jean
Gouranton, and
Isabelle
Pellerin
From the UPRES-A CNRS 6026, Biologie Cellulaire et Reproduction,
Equipe Canaux et Récepteurs Membranaires, Université de
Rennes 1, Campus de Beaulieu, Bâtiment 13, 35042 Rennes
cedex, France
 |
ABSTRACT |
The MIP (major intrinsic protein) proteins
constitute a channel family of currently 150 members that have been
identified in cell membranes of organisms ranging from bacteria to
man. Among these proteins, two functionally distinct
subgroups are characterized: aquaporins that allow specific water
transfer and glycerol channels that are involved in glycerol and small
neutral solutes transport. Since the flow of small molecules across
cell membranes is vital for every living organism, the study of such
proteins is of particular interest. For instance, aquaporins located in
kidney cell membranes are responsible for reabsorption of 150 liters of
water/day in adult human. To understand the molecular mechanisms of
solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located
at the same positions in glycerol channels. Here, we show that
substitution of a tyrosine and a tryptophan by a proline and a leucine,
respectively, in the sixth transmembrane helix of an aquaporin leads to
a switch in the selectivity of the channel, from water to glycerol.
 |
INTRODUCTION |
Based on amino acid sequence, members of the
MIP1 family are predicted to
share a common topology consisting in 6 transmembrane domains connected
by 5 loops (A-E). From biochemical and biophysical data, a model
representing these proteins as "hourglasses" has been proposed (1)
(Fig. 1A). In this model, the
channel pore is constituted by the junction of loops B and E that
overlap midway between the leaflets of the membrane. Recently, the
three-dimensional structure of the first identified aquaporin, AQP1
(2), has been obtained and has defined that the protein complex is
constituted by four monomers (3-5). Each monomer is formed by six
tilted
helices spanning the membrane bilayer and surrounding a
central density zone. This zone represents likely the narrowest segment of the water pore and may be constituted by loops B and E according to
the hourglass model. As opposed to the increasing amount of data aiming
to determine aquaporins structure, no study concerning glycerol
channels has been reported, but considering their high level of
identity, it was assumed that they had the same structural organization. Using a biochemical approach, we showed recently that an
insect aquaporin, AQPcic (6), is tetrameric in cell membrane, like
AQP1, whereas the glycerol channel of Escherichia coli
(GlpF) is a monomer (7). These results suggest that oligomerization of
MIP proteins could be involved in transport selectivity. In order to
elucidate molecular mechanisms that are accountable of the channel
selectivity, we have developed a strategy consisting in a systematic
comparison of the physico-chemical properties of amino acids at each
position in multiple sequence alignments (8). We have identified five
positions (P1-P5) corresponding to amino acid residues conserved in
aquaporins and glycerol channels but with highly different
physico-chemical properties in the two subgroups. Interestingly, four
positions (P2, P3, P4, P5) highlighted by our sequence analysis are
located in, or very close to, loop E, strengthening the idea that this
loop is involved in the pore structure and/or selectivity of the
protein. In this paper, we performed a functional study combined to an
oligomerization state analysis of AQPcic mutants. We demonstrate that
substitution of a tyrosine and a tryptophan by a proline and a leucine,
respectively, on positions P4P5 of AQPcic abolishes the water transfer
and allow the glycerol passage through the protein.

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Fig. 1.
Topological model of MIP proteins (1).
A, the six bilayer spanning domains (helices 1 to 6), loops
A-E, the two NPA motifs (Asn-Pro-Ala), and positions P1, P2, P3, P4,
P5 are indicated. B, sequence alignment of the region
encompassing positions P2, P3, P4, P5 in AQPcic and GlpF.
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EXPERIMENTAL PROCEDURES |
Plasmids Construction and Mutagenesis--
The pSP-AQPcic vector
corresponds to the full-length AQPcic coding sequence inserted into the
pX
G-ev1 plasmid (6). The AQPcic mutants were obtained by performing
a two-step reaction polymerase chain reaction with sets of appropriate
primers overlapping in the region of the mutation (9). The mutated
AQPcic cDNA were cloned either in pX
G-ev1 (constructs were
termed pSP-AQPS205D, pSP-AQPA209K, and
pSP-AQPcicY222P/W223L) or in the yeast expression vector
pYeDp60 (pY60-AQPcic and pY60-AQPcicY222P/W223L).
The pSP-glpF vector has been obtained by cloning the entire coding
region of E. coli glpF (from the pglpF vector, generously given by Dr. Mizumo (10)) into the BglII site of the
pX
G-ev1 plasmid.
AQPcic and GlpF Expression in Xenopus Oocytes--
Wild type or
mutated cRNA were prepared in vitro with the mRNA
capping kit (Stratagene) using either pSP-AQPcic,
pSP-AQPS205D, pSP-AQPA209K, and
pSP-AQPcicY222P/W223L or pSP-glpF as templates. The cRNA
were injected into stages VI Xenopus oocytes and osmotic water or apparent glycerol permeabilities of oocytes were measured as
described in Ref. 6. The [14C]glycerol uptake assays were
performed as described in Ref. 11.
Oligomerization State and Protein
Analysis--
Xenopus total membranes were prepared as
described in Ref. 12. Yeast or Xenopus overexpressed
proteins were extracted and analyzed on a linear 2-20% sucrose
density gradient as described previously (7, 13). Briefly, the protein
extracts were submitted to an ultracentrifugation to equilibrium at
100,000 × g for 16 h (5 °C), and the fractions
were collected. Xenopus protein extracts or gradient
fractions were loaded on SDS gel (14) and electrotransferred onto
polyvinylidene difluoride filters (15). Immunodetections were performed
using polyclonal rabbit antisera raised against the native AQPcic (16)
or raised against a synthetic C-terminal peptide of GlpF (7).
 |
RESULTS |
We have constructed mutants of AQPcic by substituting
characteristic amino acids of aquaporins with corresponding glycerol channels amino acids at positions P2 (AQPcic-S205D), P3 (AQPcic-A209K), and P4P5 (AQPcic-Y222P/W223L). Transcripts corresponding to AQPcic and
mutants AQPcic and GlpF were injected into Xenopus oocytes, and the presence of proteins in the oocyte membranes was verified by
Western blot (Fig. 2).

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Fig. 2.
Expression of AQPcic, mutant AQPcic, and GlpF
in oocytes. Western blots were performed with total protein
membrane extracts of Xenopus oocytes injected with AQPcic
cRNA (AQPcic), mutant AQPcic cRNA (S205D, A209K, Y222P/W223L), GlpF
cRNA (GlpF), or H2O (control). Immunodetections were
achieved with anti-AQPcic antibodies (A) or with antibodies
directed against a synthetic peptide of GlpF (B).
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Functional assays were achieved by measuring the water permeability
(Pf), the apparent glycerol permeability
(P'gly), and the glycerol uptake of oocytes
expressing the different proteins. As shown previously, oocytes
injected with AQPcic cRNA present a 14-fold increase of
Pf compared with control oocytes (Fig.
3A). In contrast, oocytes
injected with mutants AQPcic (AQPcic-S205D, AQPcic-A209K, and
AQPcic-Y222P/W223L) or GlpF cRNA do not demonstrate any
Pf increase (Fig. 3A). In Fig.
3B, apparent glycerol permeability is evaluated for each
oocyte batch. Remarkably, oocytes expressing AQPcic-Y222P/W223L behave
like GlpF expressing oocytes and exhibit a 8-fold increase of
P'gly compared with control oocytes. When injected with GlpF cRNA, oocytes exhibit a 9-fold increase of P'gly, whereas in oocytes expressing AQPcic,
AQPcic-S205D, or AQPcic-A209K, no modification of
P'gly is observed as compared with controls. To
reinforce these data, an experiment of glycerol uptake has been
performed using [14C]glycerol. The results confirm a
significant increase in glycerol uptake of 9-fold for oocytes
expressing AQPcic-Y222P/W223L and of 8-fold for GlpF expressing oocytes
as compared with controls (Fig. 3C).

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Fig. 3.
The mutant AQPcic-Y222P/W223L
loses its ability to transport water and acquires glycerol
transport properties. For each experiment,
Pf, P'gly, and glycerol
uptake were measured from 10 oocytes expressing either AQPcic
(AQPcic), AQPcic-S205D (S205D), AQPcic-A209K
(A209K), AQPcic-Y222P/W223L (YW222/223PL), or
from H2O-injected oocytes (Control). The given
Pf (A) and
P'gly (B) are the average of five
independent experiments. Glycerol uptake (C) is given from
three experiments (values ± S.D.). Differences between means were
detected by Student's t test (*, p < 0.001 versus control).
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In parallel to swelling assays, the oligomeric organization of
AQPcic-Y222P/W223L was analyzed by sucrose gradient sedimentation. The
experiments were performed using proteins overexpressed in yeast cells
and extracted either with denaturing detergent (SDS, sodium dodecyl
sulfate) or nondenaturing detergent (OG,
n-octyl-
-D-glucopyranoside). As shown in Fig.
4, AQPcic extracted with OG peaks at
sedimentation fraction number 9-10. These fractions correspond to a
6.8 S apparent sedimentation coefficient mean value that fits with a
homotetrameric form of the protein. When protein solubilization is
performed with SDS, sedimentation occurs at fraction numbers 14 and 15 (mean value of 2.8 S) that correspond to the monomeric form of the
protein. In opposition, the mutant AQPcic-Y222P/W223L constantly peaks at fractions corresponding to a monomer whatever the detergent used for
the solubilization of the proteins and thus behaves like the GlpF
protein (Fig. 4).

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Fig. 4.
The mutant AQPcic-Y222P/W223L is a
monomer. AQPcic and AQPcic-Y222P/W223L were extracted from yeast
membranes and GlpF from Xenopus total membranes. Proteins
were extracted with a nondenaturing detergent (OG) or a
denaturing detergent (SDS) and then analyzed on sucrose
gradient. Positions of marker proteins cytochrome C (CytC,
1.7 S), bovine serum albumin (BSA, 4.3 S), and IgG (7 S)
detected by Coomassie Blue staining of acrylamide gels are indicated at
the top. AQPcic is tetrameric in OG (6.8 S), whereas
AQPcic-Y222P/W223L is a monomer (2.8 S).
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DISCUSSION |
Accumulating data indicate that MIP proteins contain two major
physiological subgroups. Recently, we (8) and others have reported that
the separation of these two subfamilies might be based on few sequence
and/or motif distinctions (17-19). In particular, we have identified
five positions where amino acids are drastically different in the two
subfamilies. Besides aquaporins that are only permeable to water, some
homologues are permeated by water, glycerol, and other small solutes:
AQP3 identified in kidney (20-22), AQP7 isolated from rat testis (23),
and the liver AQP9 (24) belong to this group referred as
aquaglyceroporins. Thus, the puzzling question that currently emerged
is how the specificity of proteins for water and/or small solutes is
achieved. Our data reveal that substitution of amino acids on
aquaporins by corresponding glycerol facilitator residues at positions
P2, P3, P4P5 abolishes water transport, demonstrating the crucial role
of these amino acids in aquaporin function. Furthermore, the AQPcic
mutant that has been substituted on positions P4P5 by glycerol channels
amino acids gains competence to transport glycerol. Oligomerization analysis further demonstrates that this mutant loses its ability to
form a tetramer. The involvement of these two conserved amino acids
positions in both oligomerization and specificity of the proteins gives
a new important parameter that need to be considered for structural and
functional studies on MIP proteins. Indeed, in addition to amino acids
strictly involved in the pore structure, one should also take into
account amino acids that are necessary for oligomer assembly, without
excluding the fact that some residues might be implicated in both
mechanisms. Positions P4P5 are located at the top of helix 6 in MIP
proteins and correspond to aromatic residues in aquaporins whereas a
proline followed by a non aromatic residue is always found in the
glycerol channels group (Fig. 1B). The role of proline in
determining local conformation in proteins is well known (25). The
presence or absence of such residue in MIP proteins provokes
undoubtedly a different conformation of helix 6 and/or influences
regions closed to the helix. Since loop E, that is supposed to
participate in the pore of the protein, is very close to this helix,
the interaction between the loop E and helix 6 may modulates the
aperture of the pore and thus be responsible of the protein
specificity. Using the three-dimensional model (3) of AQP1, Walz
et al. (5) have assigned each helix of the protein by their
corresponding numbers in the hourglass model. According to their
analysis, each monomer of AQP1 interacts with each other by contacts
between helices 4 and 6. Conserved amino acids located in these helices
are thus essential to maintain the tetrameric structure of aquaporins.
This structural organization of aquaporin monomers explains the crucial
role of amino acids at positions P4P5 in the conformation of the
protein. In this paper, we report for the first time a double mutation
that modifies the protein structure in such a way that it allows a
change in the nature of the transported molecule from water to
glycerol. We point out two amino acids that are involved in the
selective transport by MIP proteins. We further demonstrate that the
specificity switch (water to glycerol transport) is correlated to a
change in oligomerization state of the proteins. We thus propose that oligomerization state is part of the mechanisms utilized by MIP proteins to ensure selectivity.
 |
ACKNOWLEDGEMENT |
We thank Dr. Cory Abate for her critical
review of the manuscript.
 |
FOOTNOTES |
*
This work was supported by La Fondation Langlois.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-2-99-28-61-13; Fax: 33-2-99-28-14-77; E-mail:
Isabelle.Pellerin{at}univ-rennes1.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
MIP, major intrinsic
protein;
OG, n-octyl-
-D-glucopyranoside.
 |
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