Functional analysis of aquaporin-2 mutants associated with
nephrogenic diabetes insipidus by yeast expression
Itsuki
Shinbo1,2,
Kiyohide
Fushimi1,3,
Michihiro
Kasahara4,
Kazushi
Yamauchi1,
Sei
Sasaki1, and
Fumiaki
Marumo1
1 Second Department of Internal
Medicine, 2 Department of
Physiology, and 3 Department of
Medical Informatics, Tokyo Medical and Dental University School of
Medicine, and 4 Laboratory of
Biophysics, School of Medicine, Teikyo University, Tokyo 113-8519, Japan
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ABSTRACT |
Mutations of aquaporin-2 (AQP2) vasopressin water channel cause
nephrogenic diabetes insipidus (NDI). It has been suggested that
impaired routing of AQP2 mutants to the plasma membrane causes the
disease; however, no determinations have been made of
mutation-induced alterations of AQP2 channel water
permeability. To address this issue, a series of AQP2 mutants were
expressed in yeast, and the osmotic water permeability
(Pf) of the
isolated vesicles was measured. Wild-type and mutant AQP2 were
expressed equally well in vesicles. Pf of the
vesicles containing wild-type AQP2 was 22 times greater than that of
the control, which was sensitive to mercury and weakly dependent on the
temperature. Pf
measurements and mercury inhibition examinations suggested that mutants
L22V and P262L are fully functional, whereas mutants N68S, R187C, and
S216P are partially functional. In contrast, mutants N123D, T125M,
T126M, A147T, and C181W had very low water permeability. Our results
suggest that the structure between the third and fifth hydrophilic
loops is critical for the functional integrity of the AQP2 water
channel and that disruption of AQP2 water permeability by mutations may
cause NDI.
water channel; vasopressin
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INTRODUCTION |
AQUAPORIN-2 (AQP2) is a member of the aquaporin
water channel family (1), which is a group of ancient membrane
intrinsic proteins (10) involved in water transport (16, 19). AQP2 is
designated as the vasopressin water channel, because the
vasopressin-stimulated translocation of AQP2 to the apical membrane is
needed for regulatory water reabsorption and urine concentration (36,
45). The importance of this protein has been highlighted by the
identification of mutations in the AQP2 gene in patients with
nephrogenic diabetes insipidus (NDI), a rare hereditary disease in
which the kidney cannot produce concentrated urine in response to
vasopressin (14). However, the relationship between mutation-induced
alterations in AQP2 structure and the pathogenesis of NDI is not clear.
Accelerated degradation, misrouting, and the loss of water permeability
of the AQP2 protein by mutation-induced structural changes have been postulated as the possible causes of NDI (41). Expression of various
AQP2 mutants in Xenopus oocytes has
suggested that many AQP2 mutations inhibit its proper routing to the
plasma membrane of the channel; however, the effects of these mutations
on the intrinsic AQP2 water permeability are not clear (13, 31, 32). Thus the biophysical characterization of AQP2 mutants is important for
understanding the pathogenesis of NDI.
Furthermore, functional analyses of AQP2 mutants will provide
significant insights into a classic and fundamental issue in membrane
biology, namely, how water molecules pass through a protein aqueous
pore. Many groups have investigated the structure of the aquaporins by
expressing site-directed mutants (3, 23) or NDI mutants in
Xenopus oocytes (13, 32, 41).
Aquaporins are predicted to have six transmembrane segments connected
by five hydrophilic loops. The hourglass model proposed for AQP1 structure suggested that an aqueous pore is assembled with the second
and fifth hydrophilic loops, the amino acid sequences of which are
highly conserved among the aquaporin family (23). From analyses of AQP2
structure, importance of the third and fourth hydrophilic loops for
water permeability was suggested (3). Although structural models
proposed by these studies are roughly in agreement with recent findings
by electron crystallography (9, 44), details on the structure of the
aqueous pore have been controversial and left unresolved. It has been
difficult to evaluate the channel water permeability of some of the AQP mutants using Xenopus oocytes because
of the reduced plasma membrane expression of some mutants (26, 33). On
the other hand, a yeast expression system has been used for directly
analyzing function of many transporters and their mutants, because this
system does not require intact plasma membrane expression of
transporters, which are often disturbed by protein mutation (24, 34,
39). Recently, this system has been successfully applied to study
aquaporins (12, 27) and those with mutations (26). In this study, the AQP2 mutants were expressed sufficiently well in yeast intracellular vesicles, and the intrinsic channel function of a series of AQP2 mutants was determined.
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METHODS |
Yeast strain and plasmid construction.
The protease-deficient BJ3505 strain (30) of
Saccharomyces cerevisiae was used in this study (24). The rat AQP2 mutants L22V, N68S, N123D, T125M, T126M,
A147T, C181W, R187C, S216P, and P262L were generated by PCR-based
site-directed mutagenesis. N123D and C181W have been shown to be
nonfunctional by oocyte expression study (3). All the mutants except
N123D have been shown to be related to NDI (8). In this study, a series
of rat AQP2 mutations were chosen to determine the structure of the
aqueous pore in conjunction with our previous report (3). It is
conceivable that the difference between rat and human AQP2 is not
significant for mutation analyses, because the primary structures of
rat and human AQP2 are more than 90% identical, and because rat and
human AQP2 have similar channel water permeability and mercury
sensitivity (25). The nucleotide sequences of both strands of the
mutants were verified by DNA sequence analysis using an automated
fluorescence sequencer (model 373A, Applied Biosystems). The PCR
fragments generated with two primers, 5'-CGC GGA TCC AGC ATG TGG
GAA CTC AGA TCC-3' and 5'-CGC GAA TTC TCA GGC CTT GCT GCC
GCG-3', encoding the open reading frames of wild-type rat AQP2
(WT-AQP2) and the mutants were subcloned between a GAL10-CYC1 promoter
and a phosphoglycerate kinase terminator using the
BamH I and
EcoR I sites of a yeast expression
vector, pYeDP10, which was kindly provided by Dr. Philippe (21, 37).
For a yeast expression vector, pYES2 (Invitrogen), PCR fragments
generated with two primers, 5'-CCC AAG CTT AGC ATG TGG GAA CTC
AGA TCC-3' and 5'-CCC TCT AGA TCA GGC CTT GCT GCC GCG-3', were subcloned using
Hind III and
Xba I sites downstream of a GAL1
promoter. pYeDP10 and pYES2 encode
URA3 as a selectable marker in yeast
cells. The yeast cells were transformed with the recombinant plasmids
using a lithium acetate protocol (22). The transformants were selected
by growth on plates containing synthetic minimal medium (SD) (40)
supplemented with 2% glucose and 0.08% amino acid mixture but lacking
uracil (Clontech). To induce the expression of the AQP2 mutants, single
yeast colonies were picked and grown at 30°C until they reached an
optical density at 650 nm
(OD650) of ~2-3 in SD
supplemented with 2% galactose and 0.08% amino acid mixture lacking
uracil (SD-galactose medium). From this stock, the yeast cells were
diluted to an OD650 of 0.05 into
400 ml of SD-galactose media and grown at 30°C until they reached
an OD650 of ~1.2-1.8.
Immunostaining of AQP2-expressing yeast cells indicated intracellular
distribution of AQP2 protein was similar for WT and mutants.
Vesicle preparation. A previously
described procedure (39, 43) was modified to isolate intracellular
vesicles from yeast cells. The cells were collected by centrifugation,
washed with cold 10 mM NaN3,
resuspended in 50 ml of spheroplasting media (1.4 M sucrose, 50 mM
K2HPO4,
pH 7.4, 10 mM NaN3, and 40 mM
-mercaptoethanol) containing 217 µg/ml of zymolyase 100T
(Seikagaku), and incubated at 37°C for 1 h. The spheroplasts were
collected by centrifugation (5,000 g,
5 min), suspended in 8 ml of cold hypotonic lysis buffer [0.8 M
sucrose, 10 mM triethanolamine, pH 7.2, 1 mM EDTA supplemented with
protease inhibitor cocktail tablets (Boehringer Mannheim)] and
lysed with 20-30 strokes of a glass Dounce homogenizer on ice. The
homogenate was centrifuged at 3,000 g
for 10 min at 4°C, and the sucrose concentration of the supernatant
was adjusted to 1.4 M. A discontinuous sucrose gradient [2 M (1.5 ml), 1.6 M (3 ml), 1.4 M (6 ml, containing homogenate), and 0.8 M
sucrose (1.5 ml)] was centrifuged for 3 h at 100,000 g in an SW41 rotor (Beckman) at
4°C. Following centrifugation, three interfacial layer fractions
were collected, and each was washed twice (95,000 g, 20 min) with 2 ml of ice-cold
vesicle buffer (50 mM mannitol, 90 mM KCl, 1 mM EDTA, and 20 mM
Tris · HCl, pH 7.4) and stored on ice until further use.
Immunoblot analysis. SDS-PAGE was
performed as described (18). The total cell lysates and vesicle
fractions were incubated with SDS sample buffer [1.5% SDS, 30 mM
Tris · HCl, pH 6.8, 2.5%
-mercaptoethanol, and
5% (vol/vol) glycerol] at 37°C for 1 h. A 20-µl sample was
loaded in each lane, separated by a 10-20% continuous-gradient gel for 1 h with a 40-mA current, and
electrotransferred to an enhanced chemiluminescence (ECL)
nitrocellulose membrane (Amersham Pharmacia Biotech). After the
membrane was blocked with Superblock (Promega) for 1 h at 4°C and
washed once with TBS-T (20 mM Tris, 150 mM NaCl, and 0.05% Tween-20,
pH 7.4), it was incubated with affinity-purified anti-AQP2 antibody
against a synthetic peptide corresponding to 15 carboxy-terminal amino
acid residues of rat AQP2 (19) diluted at 1:1,000 in TBS-T at room temperature for 1 h, then washed three times with TBS-T. Next, the
membrane was incubated with biotin-labeled anti-rabbit IgG antibody
(Vector, Burlingame, CA) diluted 1:500 in TBS-T at room temperature for
1 h, washed three times, and incubated with a 1:500 dilution of the ABC
mixture (Vector) at room temperature for 30 min. After the blot was
washed with TBS-T three times, the blot was visualized by ECL using an
ECL mini camera (Amersham Pharmacia Biotech). For
125I detection, after the
incubation with primary antibody, the washed membrane was reacted with
3 µCi of
125I-labeled protein A (Amersham
Pharmacia Biotech) in TBS-T for 1 h, washed again, exposed to X-ray
film for 4 h, and developed (24).
Vesicle osmotic water permeability
measurement. The osmotic water permeability
(Pf) of the
yeast vesicles was measured by a light-scattering method using a
SX-18MV stop-flow apparatus (Applied Photophysics, Leatherhead, UK).
The instrument dead time was 1.6 ms, and sample temperature was
controlled by circulating water bath. The vesicles were diluted at 1:30
with the vesicle buffer. The vesicles suspension was mixed abruptly
with the same volume of the vesicle buffer containing 360 mM mannitol,
and 180 mM inwardly directed osmotic gradient was imposed. A decrease in the cell volume due to osmotic water efflux driven by the osmotic gradient was monitored as the time-dependent increase in the 90° scattered light intensity monitored at 466 nm. A light intensity trace
was obtained for each sample by averaging four to seven measurements.
The light scattering intensities were fitted to biexponential curves,
and Pf was
calculated as described (2, 17) by iteratively solving the following
equation with Mathematica software (Wolfram Research, Champaign, IL)
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where
V(t) is the volume of the cell at
time t, SAV is the surface
area-to-volume ratio at t = 0 (2.58 × 105
cm
1, calculated from the
average vesicle diameter of 233 nm measured by a Coulter counter), vw
is the molecular volume of water (18 cm3/mol),
Osmin is the osmolarity inside the
vesicles, and Osmout is the
osmolarity outside the vesicles.
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RESULTS |
Expression of AQP2 in yeast cells. The
expression of AQP2 in a protease-deficient yeast strain BJ3505 was
induced by switching cells from glucose- to galactose-containing media.
No immunoreactive band was observed in native yeast vesicles or in
vesicles from mock-transfected yeast cells, indicating that no protein
that cross-reacts with AQP2 antibody is present in BJ3505 cells. In addition, no band was observed in total cell lysate prepared from yeast
cells transfected with the AQP2 vector incubated in glucose medium
(Fig.
1A).
After stimulation by glucose depletion and galactose supplementation,
AQP2 expression was induced, and it accumulated in yeast cells in a
time-dependent manner. A large amount of AQP2 had accumulated by mid-
and late-exponential growth phase between 12 and 18 h galactose
induction after preincubation in the galactose medium (Fig.
1A, lanes
4 and 5). The
apparent molecular mass of the AQP2 expressed in the yeast cells was
~29 kDa, identical to that in
LLC-PK1 cells (Fig.
1B). The bands with a molecular mass of ~60 kDa, which were occasionally observed in both yeast cells and
LLC-PK1 cells, are likely to be a
dimer of AQP2. These are unlikely to be N-glycosylated forms of
AQP2, since AQP2 is not N-glycosylated in yeast cells (12) or
in LLC-PK1 cells (17). Although
native AQP2 is partly N-glycosylated (18),
N-glycosylation is not necessary for the integrity of the water
permeability function of AQP2 (3). These results indicate that the AQP2
protein is properly processed and expressed in yeast cells, similar to
that in mammalian cells. Note that no significant degradation products of expressed AQP2 were observed in yeast cells, which is helpful for
generating the maximum amount of functional product and for analyzing
channel water permeability of AQP2 and the mutants without potential
interference from the degradation products. No significant difference
was found between the two yeast expression vectors, pYES2 and pYeDP10.
In the subsequent studies, pYeDP10 was used as the yeast expression
vector, and the cells were harvested at mid- and late-exponential
growth phase after 12-18 h of galactose incubation.

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Fig. 1.
A: effects of induction conditions on
heterologous aquaporin-2 (AQP2) expression in yeast cells. Total cell
lysates prepared as described (24) were isolated from yeast cells, and
15 µg of protein was applied to each lane, separated by SDS-PAGE, and
blotted. Lane 1, 24-h glucose culture;
lane 2, 12-h galactose culture after
24-h glucose culture; lane 3, 24-h
galactose culture; lane 4, 12-h
galactose culture; and lane 5, 18-h
galactose culture after preculture in galactose. Molecular mass
standards (kDa) are indicated on the
left.
B: expression of AQP2 protein in yeast
cells using two different yeast expression vectors, pYES2 and pYeDP10.
Fifteen micrograms of protein was obtained from total cell lysates of
LLC-PK1 cells
(lane 1) and yeast cells
(lanes 2-5), separated by
SDS-PAGE, and blotted. Lane 1,
wild-type (WT) AQP2-transfected
LLC-PK1 cells;
lane 2, pYES2 vector;
lane 3, pYeDP10 vector;
lane 4, pYE-AQP2; and
lane 5, pYeDP10-AQP2. Molecular mass
standards (kDa) are indicated on the
left.
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Osmotic water permeability of yeast vesicles
expressing AQP2. Yeast cell homogenates were
fractionated by a discontinuous sucrose-gradient centrifugation to
obtain fractions rich in vesicles expressing AQP2 (29, 47). The pellet
was discarded, and three interfacial layer fractions were collected.
Subsequently, immunoblot analyses and measurements of the osmotic water
permeability of each fraction were performed. For the vesicles of all
fractions from mock-transfected cells, no immunoreactive band was
detected, and the time-dependent increases in the scattered-light
intensity by stop-flow analysis were very slow (see Fig.
3B). For yeast cells expressing
WT-AQP2, immunoblot analyses indicated that AQP2 is most abundant in
the middle interfacial layer fraction between 1.4 M and 1.6 M. Vesicles
from this fraction produced rapid changes in the scattered-light
intensity by stop-flow analysis, indicating that these vesicles have
high water permeability. In the subsequent experiments, vesicles from
the middle fraction were used. Size distribution analysis of the
vesicles determined by quasi-elastic light scattering indicated that
the vesicles consisted of a single population with a mean diameter of
233 ± 15 nm (Fig. 2). The diameters of
the vesicles from the yeast transfected with mock plasmid, WT-AQP2, and
AQP2 mutants were not significantly different.

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Fig. 2.
Measurements of yeast vesicle size by quasi-elastic light scattering.
Yeast vesicle diameter was determined by quasi-elastic light scattering
with a Coulter counter N4 Plus. Ordinate represents the fraction of the
vesicle population. Abscissa represents the vesicle diameter.
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Figure 3 shows representative traces of
vesicle shrinkage after the imposition of a 180 mOsm inwardly directed
osmotic gradient. The vesicles were isolated from WT-AQP2- and
mock-transfected yeast cells. Vesicle volume decreased ~63% in both
vesicles, and vesicle shrinkage correlated linearly with the increase
in the osmolality up to +230 mOsm (data not shown), indicating that the change of the vesicle volume perfectly reflects the change of osmolarity inside the vesicles under our experimental condition. Vesicles from mock-transfected yeast exhibited very low osmotic water
permeability, suggesting that this yeast expression system is suitable
for the heterologous expression of aquaporin water channels (Fig.
3B). Although a few members of the
MIP family are present in yeast (4, 28, 38), functional water channels were not found normally present in BJ3505 yeast vesicles. The osmotic
water permeability
(Pf) of
vesicles expressing WT-AQP2 was 328 ± 19 µm/s (mean ± SE,
n = 9), which was 22-fold greater than
that of mock-transfected vesicles (15 ± 2 µm/s,
n = 8). Two additional characteristics
of the AQP2 water channel, that is, its inhibition by mercury compounds
and its weak temperature dependence, were examined. The
Pf of vesicles
expressing WT-AQP2 after treatment with 0.3 mM
p-chloromercuribenzoic acid (pCMB) was
nearly identical to that of the mock-transfected control (14 ± 1 µm/s, n = 4) (see Fig.
7A). Figure
4 shows an Arrhenius plot for the
temperature dependence of the osmotic water permeability. The
activation energy (Ea), given by
the slope of the Arrhenius plot, was 2.0 ± 0.2 kcal/mol for
AQP2-expressing vesicles and 13.1 ± 0.5 kcal/mol for
mock-transfected yeast vesicles. These data indicate that functional
AQP2 proteins are efficiently expressed in yeast vesicles.

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Fig. 3.
Osmotic water permeability of AQP2-containing yeast vesicles. Stop-flow
measurements of osmotic water permeability of vesicles isolated from
AQP2-transfected yeast cells (A) and
mock-transfected yeast cells (B) at
10°C. Representative traces and curves fitted to a biexponential
are shown. Inset: an early portion of
the time course showing rapid vesicle shrinking.
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Fig. 4.
An Arrhenius plot for the temperature dependence of osmotic water
permeability in vesicles isolated from the mock-transfected ( ) and
AQP2-transfected yeast cells ( ). Each data point is the average of
4-7 measurements. Fitted line slopes yielded activation energies
of 13.1 ± 0.5 kcal/mol for the control and 2.0 ± 0.2 kcal/mol
for WT-AQP2.
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Osmotic water permeability of AQP2 mutants causing
NDI. The 10 AQP2 mutants were expressed in yeast cells,
and the vesicles from these cells were isolated. The expression of AQP2
mutant proteins was examined by immunoblot analysis. The quantity and the molecular weight of the expressed AQP2 mutants were almost identical to those of WT-AQP2, indicating that AQP2 mutants were synthesized and processed similar to that of WT-AQP2 in yeast cells
(Fig. 5). The slightly larger apparent
molecular weight of N123D and S216P may be due to residual differences
in the molecular structure even with SDS denaturation or in the protein
modification of these mutants as observed in
LLC-PK1 cells (5, 11). Similar levels of protein expression of WT and mutant AQP2 in
yeast vesicles confirmed that the yeast expression system is suitable
for the direct biophysical characterization of AQP2 mutants. The
Pf of vesicles
expressing each type of mutant was measured (Fig.
6), and the data are summarized in Fig.
7A. Single
channel water permeabilities of AQP2 mutants relative to that of WT
estimated roughly by calculating Pf values per
unit protein expression determined from immunoblot (Fig. 5) were 0.88 (L22V), 0.58 (N68S), 0.05 (N123D), 0.07 (T125M), 0.04 (T126M), 0.08 (A147T), 0.05 (C181W), 0.31 (R187C), 0.44 (S216P), and 0.71 (P262L). In
the absence of pCMB, L22V and P262L yielded a very high
Pf, similar to
that of WT. The
Pf of vesicles
expressing N68S, R187C, and S216P was higher than that of the
mock-transfected control vesicles but significantly lower than that of
WT. The Pf of
these mutants was inhibited by pCMB, indicating that these mutants are
functional water channels. In contrast, the
Pf of vesicles
expressing N123D, T126M, A147T, and C181W was very low and similar to
that of the control vesicles. Thus the AQP2 mutants examined in this
study can be classified into three groups: nonfunctional mutants
(N123D, T126M, A147T, C181W), partially functional mutants (N68S,
R187C, S216P), and fully functional mutants (L22V, P262L). A comparison
of the location and the channel water permeability of these mutants
suggests that the position of the missense mutation is a crucial
determinant for its effects upon water channel function (Fig.
7B). Mutations between the third and
fifth hydrophilic loops appear to disrupt water channel function,
whereas mutations closer to the termini have less drastic effects on
function.

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Fig. 5.
Expression of nephrogenic diabetes insipidus (NDI)-causing AQP2 mutants
in yeast vesicles by immunoblot analysis. Vesicle fractions were
isolated from yeast cells expressing AQP2 mutants, and 5 µg of
protein was applied to each lane. Each sample was separated by SDS-PAGE
and blotted. Arrow indicates the position of the 29-kDa core protein of
WT-AQP2.
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Fig. 6.
Comparison of time-dependent volume decreases after the osmotic shock
of vesicles expressing a series of AQP2 mutants. A 180 mOsm inwardly
directed osmotic gradient was imposed on vesicles isolated from yeast
cells expressing AQP2 mutants, and subsequent vesicle shrinkage was
observed by a stop-flow apparatus. Traces were fitted to biexponential
curves. Note the rapid decrease in vesicle volume for WT and P262L in
contrast to that for C181W and mock-transfected cells, indicating high
osmotic water permeability of WT and P262L and low permeability of
C181W and mock-transfected cells.
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Fig. 7.
A: osmotic water permeability and
mercury inhibition of yeast vesicles expressing NDI-causing AQP2
mutants. Osmotic water permeabilities
(Pf) are shown
with open bars. Effects of treatments with 0.3 mM
p-chloromercuribenzoic acid (pCMB) for
15 min were examined for samples that showed a significantly high
osmotic water permeability and are shown with hatched bars. Values are
means and SE of 8-10 experiments.
B: localization of the missense
mutations examined in this study and a summary view of our findings on
the schematic representation of AQP2. Positions and water channel
function of the AQP2 mutants are indicated, with solid circles for the
nonfunctioning mutants, hatched circles for the partially functioning
mutants, and open circles for the fully functioning mutants. Two open
boxes indicate the positions of the NPA motifs. Five hydrophilic loops
are indicated as I-V.
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DISCUSSION |
In this study, we performed the first direct characterization of
channel water permeability of NDI-related AQP2 mutants by measuring the
osmotic water permeability of yeast intracellular vesicles that express
a series of AQP2 mutants. The expression of WT-AQP2 protein in yeast
vesicles induced osmotic water permeability more than 20 times greater
than that of mock-transfected vesicles or native yeast vesicles, and
the functional properties of the water channels generated were
identical to those of AQP2 expressed in oocytes and cultured cells. We
found that this yeast expression system is suitable for quantitative
analysis of the effects of mutations on channel water permeability. In
addition, since WT and mutant AQP2 were expressed equally well in
vesicles, the channel water permeability of the expressed mutants can
be compared directly without the possibly complicating effects of
mutations on the intracellular distribution of the protein. It has been
very difficult to exclude the effects of plasma membrane expression in
the analyses of AQP2 mutations with the oocyte expression system (13,
32, 33). This yeast expression system enabled us to analyze directly and precisely the effects of various amino acid substitutions in the
AQP2 protein on channel water permeability.
We found that all of the tested mutations of AQP2 between the second
and sixth transmembrane segments have significant deleterious effects
on water channel function. The mutations N123D, T125M, T126M, A147T,
and C181W, which are between the third hydrophilic loop and the second
NPA motif, seemed to have the most disruptive effects on the water
channel function of AQP2. These results are compatible with previous
findings by oocyte experiments that N123D, T125M, and C181W are
nonfunctional water channels (3, 20). No function of A147T has been
shown in LLC-PK1 cells (46).
However, our data argue with oocyte findings that T126M and A147T have some defect in surface expression but have intact water permeability (32, 41). Possible reasons for the difference are first that water
permeability of the mutants was calculated indirectly from the ratio of
small increase in water permeability and small amount of surface
protein in oocyte experiments. Estimation of small change by ratio
calculation may often be ambiguous. In contrast, water permeabilities
of the mutants can be determined directly in our yeast vesicle
examination. Mutations do not alter AQP2 expression or protein routing
in the yeast expression system. Second, findings in oocytes are not
always relevant to other cells, as observed for CFTR
508, probably
because low temperature for oocyte culture may affect protein folding
condition (15). Thus, for mutation analyses, observations only in
oocyte expression system may be misleading.
In contrast, N68S and R187C near the two NPA motifs and S216P in the
sixth transmembrane segment had only a partially negative effect on the
channel water permeability of AQP2. Our data are again inconsistent
with previous oocyte experiments that showed that these mutants are not
functional. The difference can be attributed to the difference in
sensitivity of water permeability measurement. In yeast system,
Pf of WT-AQP2 was
as much as 20 times as large as the control but the ratio
was only about 5 for the oocyte system (41). In addition, possible
effects of high mannose glycosylation of these mutants when expressed
in oocytes were not examined. A decrease in the electrophoretical
mobility of the S216P mutant in the immunoblot indicates alterations in
the channel structure or glycosylation, but this mutation did not seem
to significantly change the function of the aqueous pore. The findings
that L22V and P262L had function nearly identical to WT-AQP2 was
compatible with previous observations in oocyte experiments (8, 31), indicating that the first transmembrane segment and the amino- and
carboxy-terminal domains are not important for the assembly of the
aqueous pore. Rather, it has been suggested that mutations in the
terminal domains may interfere with the interactions between the AQP2
protein and vesicular or cytoskeletal proteins that regulate the
intracellular trafficking of AQP2 (6, 7, 35, 42). The finding that the
regulatory exocytosis of AQP2 is controlled by the phosphorylation of
serine-256 (17) suggests that mutations near this residue may cause
defects in vasopressin-responsive channel translocation.
The channel water permeability of the AQP2 mutants in this yeast
expression system strongly supports our model of AQP2 structure proposed on the basis of oocyte expression data which demonstrates that
mutations in the third and fourth hydrophilic loops impair channel
function (3). In this model, we have emphasized the critical roles of these domains in the formation of the aqueous pore
and for the integrity of AQP2 function. Our data in this study further
validate our AQP2 structure model, because analyses of new mutations
also suggested the significance of the third and fourth hydrophilic
loops, and because examinations completely eliminated of the effects of
mutation-related misrouting are fully compatible with findings in
previous oocyte experiments. The lack of function of C181W and the
partial effects of N68S and R187C mutations in this study partially
support the hourglass model proposed from AQP1 mutation analyses (23).
These are also in accord with our model, in which the importance of the
NPA domains was proposed. Although differences in the mercury-sensitive
site among members of the aquaporin family (26, 33) and differences in
the sensitivity of osmotic water permeability to mercury between AQP1
and AQP2 have suggested that there are structural differences in the
aqueous pore of aquaporins, our studies have underscored the importance
of the structure between the third and fifth hydrophilic loops at least
for AQP2.
The presented data have confirmed that the disruption of the intrinsic
function of AQP2 by missense mutations may be a significant pathogenic
cause of NDI. It has been suggested from experimental observations with
oocytes that accelerated degradation and the inappropriate targeting of
the AQP2 mutants are the major causes of NDI (13, 32, 41). The
mechanisms by which gene mutations disrupt the function of the protein
product include a decrease in protein synthesis, disorders in
posttranslation modification including glycosylation and
phosphorylation, accelerated protein degradation, mistargeting of the
protein, and alterations in the intrinsic activity of the protein. For
AQP2, mutation-related disruptions affecting regulatory redistribution
may be potential causes of NDI. Previous studies using NDI-related AQP2
mutants expressed in Xenopus oocytes
showed that AQP2 mutants other than L22V (8) and E258K (31) are
retained in the endoplasmic reticulum and are less stable than WT-AQP2
(41). However, because the trafficking mechanisms of
channel proteins in oocytes may be different from those in mammalian
cells and there have been no studies using mammalian epithelial cells,
it cannot be concluded that the misrouting of AQP2 mutants is mainly
responsible for the pathogenesis of NDI.
In conclusion, we have directly evaluated channel water permeability of
a series of AQP2 mutants that are related to NDI without observing the
effects of intracellular misrouting. Mutations between the third and the fifth hydrophilic loops are shown to impair water
permeability, implicating the direct association of mutation-induced loss of channel water permeability and the pathogenesis of NDI. In
addition, our observations have stressed the importance of the
structure between the third and fifth hydrophilic loops for water
permeability function, confirming our structure model for AQP2.
 |
ACKNOWLEDGEMENTS |
We are grateful to Prof. Kotaro Kamino (Department of Physiology,
Tokyo Medical and Dental University School of Medicine) for valuable
discussions on this work. We thank Dr. Yumi Yamashita for technical
support. I. Shinbo is grateful to Prof. Kotaro Kamino for kind
permission to work in Second Department of Internal Medicine.
 |
FOOTNOTES |
This work was supported in part by a Grant-in-Aid for Scientific
Research A and C from the Ministry of Education, Science, Sports and
Culture, and by a Grant-in-Aid from the Japan Medical Association.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. Fushimi,
Second Dept. of Internal Medicine, Tokyo Medical and Dental Univ.
School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo
113-8519, Japan.
Received 22 December 1998; accepted in final form 22 June 1999.
 |
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