1 Second Department of Internal Medicine, A genome project focusing
on the nematode Caenorhabditis elegans has demonstrated the
presence of eight cDNAs belonging to the major intrinsic protein
superfamily. We functionally characterized one of these cDNAs named
C01G6.1. Injection of C01G6.1 cRNA increased the osmotic water
permeability (Pf) of Xenopus
oocytes 11-fold and the urea permeability 4.5-fold but failed to
increase the glycerol permeability. It has been speculated that the MIP
family may be separated into two large subfamilies based on the
presence or absence of two segments of extra amino acid residues (~15
amino acids) at the second and third extracellular loops. Because
C01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with those
of AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that of
wild-type AQP-CE1, although the values of Pf and
urea permeability were decreased by 39-74% and 28-65%,
respectively. These results suggest that the two segments of extra
amino acid residues may not contribute to channel selectivity or
formation of the route for small solutes.
aquaporin; glycerol facilitator; water permeability; glycerol
permeability; urea permeability; major intrinsic protein; Caenorhabditis elegans
WATER MOVEMENT across the plasma cell
membrane is a fundamental process for the maintenance of the
intracellular environment. Aquaporins (AQPs) are a family of integral
membrane proteins that transport water. AQPs are members of the major
intrinsic protein (MIP) superfamily and are found throughout nature
(reviewed in Refs. 7 and 18). In Escherichia coli, there are
two MIP family proteins, namely, glycerol facilitator (GlpF) (15) and
water channel (AQPZ) (1). Yeast (Saccharomyces cerevisiae)
has four MIP proteins in its genome. Plants have many MIP family
proteins; for example, there are >23 MIP family proteins in
Arabidopsis thaliana (21). Ten AQPs (AQP0-AQP9) have
been identified so far in mammals, and these are widely distributed in
water-transporting epithelia and endothelia of a variety of tissues (2,
4-6, 9, 14). In lower animals, functional water channels have been
cloned in insects (12).
The hydropathy analysis of AQP/MIP family proteins predicts six
transmembrane regions with the NH2 terminus and COOH
terminus localized in the cytosol (Fig. 1). It has been
suggested that AQP/MIP proteins may be divided into two groups,
depending on the presence or absence of longer amino acid sequences at
the second and third extracellular loops (7). In this study, we call
these segment I and segment II (Figs. 1B and
2). Previous functional expression studies have
demonstrated that AQPs lacking these segments (Fig. 1A,
e.g., AQP1, AQP2, AQP4, and AQP5) are all water selective and exclude
solutes (3, 7, 9). On the other hand, AQPs having these segments (Fig.
1B, e.g., AQP3, AQP7, and AQP9) are permeable to small
molecules such as glycerol and urea in addition to water (2, 4, 5, 15).
Another MIP protein that also has these segments, GlpF, permeates
glycerol but not water (11, 15). Thus it was previously speculated that
segments I and II participate in the formation of the transport pathway
for small solutes (2) or in the alteration of channel selectivity (7).
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (19K):
[in a new window]
Fig. 1.
Membrane topology of two types of major intrinsic protein (MIP) family
proteins (A and B). In one type
(B), extra amino acid residues (~15 amino acids) are
present at the second and third extracellular loops. These extra
segments are designated segment I and segment II in this study. In the
other type (A), these segments are absent. Both types
possess 6 presumed transmembrane segments, 5 connecting loops, and 2 conserved Asn-Pro-Ala (NPA) motifs.
View larger version (85K):
[in a new window]
Fig. 2.
Amino acid sequence of AQP-CE1 (C01G6.1) aligned with human AQP3,
glycerol facilitator protein (GlpF), human AQP1, and human AQP2. Gaps
(dashes) are inserted to maximize the matching. White letters in black
boxes denote conserved amino acid residues.
Genome projects focusing on Caenorhabditis elegans have revealed the presence of eight cDNAs encoding MIP proteins (7, 18). Of these, segments I and II are present in C01G6.1, C35A5.1, F32A5.5, and M02F4.8 and are absent in CEH09F14 and F40F9.9. K02G10.7 possesses only segment I, whereas C32C4.2 posseses only segment II. However, their functions have not yet been examined. In this study, we compared the channel function of the C01G6.1 gene product with those of AQP3 and GlpF. Moreover, we performed chimeric studies of segments I and II to determine their physiological roles.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of cDNA.
The cDNA library was constructed from mRNA of total embryo of the
wild-type nematode C. elegans directionally into
the EcoR I/Xho I site of Lambda ZAPII vector (Y. Kohara, unpublished observations). The random sequencing
of the cDNA clones identified 14 clones encoding an MIP family protein.
One clone was proved to be a cDNA reported as C01G6.1 in GenBank
(accession no. Z35595). The cDNA insert for C01G6.1 (containing the
open reading frame and untranslated sequences) was a blunt-end ligand
inserted into the Bgl II site of a pSP64T-derived Bluescript
vector containing 5'- and 3'-untranslated sequences of the -globin
gene of Xenopus (pXBG-ev1; a generous gift from Dr. Peter Agre).
In vitro cRNA synthesis and site-directed mutagenesis. Capped RNA transcripts for C01G6.1, human AQP3, and GlpF were synthesized in vitro with T3 RNA polymerase after a digestion with Not I to linearize the plasmids. Mutants of C01G6.1 were made with the PCR technique using C01G6.1 cDNA as a template (10, 11). A fragment between the Hind III and Xba I sites was replaced by a PCR fragment coding the mutants. Amino acids of segment I (DGGVRTVG) and segment II (SIFYGGAVFTK) in C01G6.1 were replaced by those in human AQP3 (ADNQLFVS and ALAGWGSAVFTTGQH, respectively) or GlpF (EQTHHIVR and WLAGWGNVAFTGGRDIP, respectively) using the mutation primers listed in Table 1. After the mutations were confirmed by a DNA sequencer (Applied Biosystems 373A), these mutant cRNAs were constructed.
|
Water, glycerol, and urea permeability of oocytes. Oocytes at stages V-VI were obtained from Xenopus laevis. Each oocyte was injected with 40 nl of water, 5 ng of wild-type or mutated C01G6.1 cRNA, human AQP3 cRNA, or GlpF cRNA and was incubated for 48 h at 20°C in Barth's buffer. The osmotic water permeability (Pf) of the oocytes was measured at 20°C from the time course of osmotic cell swelling as previously described (10). After the initial incubation, the oocytes were transferred from 200 mosM Barth's buffer to 70 mosM buffer and were imaged on a charge-coupled device camera connected to an area analyzer (Hamamatsu Photonics C3160). Serial images were stored at 0.5-s intervals in a computer. Pf was calculated from the initial 15-s response of cell swelling. To examine the effect of mercury on oocyte Pf, the oocytes were incubated in Barth's buffer containing 0.3 mM HgCl2 for 5 min before the assay. Pf was also measured at 4°C and 30°C to obtain the activation energy (Ea) from an Arrhenius plot.
The glycerol permeability (Pgly) and urea permeability (Purea) were measured from the initial rate of glycerol and urea uptake into the oocytes, respectively (8, 11). Oocytes were incubated for 5 min at 20°C in Barth's buffer containing [U-14C]glycerol (Amersham) or [14C]urea (Amersham). After the incubation, the oocytes were rapidly rinsed three times in ice-cold Barth's buffer. The individual oocytes were lysed in 0.2 ml of 10% SDS and then were subjected to liquid scintillation counting.Immunoblot analysis. Plasma membrane fractions of oocytes were obtained as previously described (19). The samples were heated at 70°C for 10 min and then separated by SDS-PAGE. Oocyte plasma membranes from 20 oocytes were applied in each lane. The samples were transferred to Immobilon-P filters (Millipore) using a semidry system. The filters were incubated for 1 h with an affinity-purified antibody against 15 COOH-terminal amino acids of the C01G6.1 gene product. The filters were further incubated for 1 h with 125I-labeled protein A solution and then examined by autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The amino acid sequence of C01G6.1 (AQP-CE1) is shown in Fig. 2. The
open reading frame encodes a protein of 290 amino acids with a relative
molecular mass calculated at 31.4 kDa. Sequence alignment clearly shows
that, just as in the case of AQP3 and GlpF, C01G6.1 also has two
segments of extra amino acid sequences (segments I and II) that are not
present in AQP1 and AQP2. No potential N-linked
glycosylation sites are present in the sequences. There is one
potential phosphorylation site by cAMP-dependent protein kinase
(residue Ser-179) and two potential phosphorylation sites by protein
kinase C (residues Ser-69 and Thr-280). A search of the protein
database revealed the highest amino acid sequence identity with AQP3
(44%) and a lesser identity with other MIP proteins, i.e., E. coli GlpF (38%), AQPZ (36%), AQP1 (34%), AQP2 (36%), AQP4
(32%), AQP5 (34%), and AQP- tonal intrinsic protein (36%).
We expressed C01G6.1, AQP3, and GlpF in Xenopus oocytes and
compared their functions. Pf of water-injected
(control) oocytes was 17 ± 2 × 104 (SE) cm/s (Fig.
3A). Injection with C01G6.1
cRNA increased Pf 10.9-fold, indicating that
C01G6.1 is a water channel (designated AQP-CE1). Similar
Pf was observed in AQP3-expressing oocytes. The
Pf of AQP-CE1-expressing oocytes was not
inhibited by incubation with 0.3 mM HgCl2, suggesting that
AQP-CE1 is a mercury-insensitive water channel.
Pf of oocytes injected with GlpF cRNA was not
different from control Pf. To examine the
temperature dependency of Pf, the
Pf was measured at 4°C and 30°C
(n = 10 each). The calculated Ea
from the Arrhenius equation of Pf was 3.9 kcal/mol, a value in the range expected for a water channel (<6
kcal/mol).
|
Next, we measured Pgly and Purea in oocytes. Pgly was not stimulated after injection of AQP-CE1 cRNA, indicating that AQP-CE1 excludes glycerol (Fig. 3A). By contrast, Pgly was increased 4.8- and 6.7-fold after injection with human AQP3 cRNA and GlpF cRNA, respectively, a finding consistent with previous reports (8, 11, 13, 15). Purea of AQP-CE1-expressing oocytes was 4.7 times higher than that of control, indicating that AQP-CE1 has a urea permeability (Fig. 3B). The Purea of AQP-CE1 was not inhibited by 0.3 mM HgCl2, as observed with Pf. The Purea of AQP3 was as high as that of AQP-CE1, whereas Purea of GlpF was similar to that of control.
To examine the role of segments I and II (Figs. 1 and 2), these segments of AQP3 and GlpF were substituted for the corresponding segments of AQP-CE1. As summarized in Fig. 4A, Pf of segment I mutants [replacement of AQP-CE1 segment I with that of AQP3 segment I (AQP3 I) or GlpF segment I (GlpF I)] was decreased by ~66-70% but was ~2.7-3.2 times higher than control Pf. Segment II mutants (AQP3 II and GlpF II) also had water channel function, although Pf was lowered by ~39-42%. In double mutants of segments I and II (AQP3 I/II and GlpF I/II), ~67-74% decreases of Pf were observed, but Pf was still ~2.4-3.1 times higher than control Pf. All these mutants failed to gain a glycerol permeability (Fig. 4B). As shown in Fig. 4C, Purea of segment I, II, and I/II mutants was ~2.3-6.0 times higher than control Purea, indicating that all these mutants were permeable to urea. The immunoblot of oocyte membrane detected bands at 27 kDa in wild-type and mutated AQP-CE1s (Fig. 5). The band intensities were similar, and no glycosylated bands were detected.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We characterized for the first time the function of a C. elegans MIP protein encoded by a cDNA named C01G6.1. Because the protein showed a water permeability when expressed in Xenopus oocytes (Fig. 3), we named it AQP-CE1. AQP-CE1 has a urea permeability in addition to its water channel function, but it does not have a glycerol permeability. AQP-CE1 is highly homologous to mammalian AQP3 (44%; Fig. 2). The phylogenetic comparison among MIP family members demonstrated that AQP3 is most related to bacterial GlpF and that AQP3 and GlpF have developed in a different branch from other AQP/MIP proteins (7, 18). This group is also characterized by the presence of segments I and II. Based on its sequence homology and the presence of segments I and II, AQP-CE1 seems to belong to this branch.
In human AQP3, we previously observed that the Tyr-to-Cys mutation at residue 212 (the position equivalent to the mercury-sensitive cysteine in AQP1 and AQP2) increased the inhibitory effect of mercury on Pf and Pgly in parallel and that the Tyr-to-Trp mutation at residue 212 decreased both Pf and Pgly to control levels (15). In addition, Ea for Pgly was 4.5 kcal/mol. Thus we concluded that water and small solutes may share a common pore in AQP3 and that the location of the pore site may be similar in AQP1, AQP2, and AQP3. In contrast, Echevarria et al. (2) previously observed no mercurial inhibition on Pgly and a high Ea for Pgly and Purea (>12 kcal/mol) in AQP3, raising the hypothesis that segments I and II contribute to a formation of the transport pathway for small solutes that is independent of the pore for water transport. We replaced segments I and II of AQP-CE1 with those of AQP3 and GlpF. If the above speculation were correct, the mutants of AQP3 I, AQP3 II, and/or AQP3 I/II could be expected to gain glycerol permeability, and the mutants of GlpF I, GlpF II, and/or GlpF I/II could be expected to lose urea permeability and gain glycerol permeability. Our results indicated that the basic characteristics of these mutants were not different from those of wild-type AQP-CE1, namely, the oocytes expressing these mutants possessed water and urea permeability but no glycerol permeability (Fig. 4). Thus segments I and II may not be directly related to the channel selectivity or to formation of the route for small solutes. However, the decreases in Pf (~39-74%) and Purea (~28-65%) were evident in these mutants, suggesting that segments I and II may play roles in the activity of the channel.
The present study and previous studies have revealed different patterns
of water and solute selectivity in AQP/MIP proteins. For example, AQP1
and AQP2 are water selective and exclude solutes (3, 9); GlpF is
glycerol selective and excludes water and urea (Fig. 3 and Ref. 15);
AQP3 and AQP7 are permeable to water, glycerol, and urea (4, 8, 11);
and AQP-CE1 and AQP9 are permeable to water and urea but
exclude glycerol (Fig. 3 and Ref. 5). If we assume that water and
solutes pass through the same pore, then we can safely conclude that
the physical pore size alone is not the determinant of the selectivity
of the pore. Recently, the three-dimensional structure of AQP1
has been partially explored (16, 20). These studies suggest
that six -helices form a central pore and that the first
intracellular loop and third extracellular loop [both contain
Asn-Pro-Ala (NPA) motifs] bend into the lipid bilayer to the center of
this pore. Strictly conserved NPA motifs seem to be indispensable for
the formation of the pore. If this model for AQP1 were found to be
applicable to other AQP/MIP proteins, it would be tempting to speculate
that amino acid sequences surrounding NPA motifs define the selectivity
of the channel. This possibility should be examined in future.
The physiological significance of water channels has not been determined in nematodes. In situ hybridization of C. elegans embryo demonstrated transient expression of AQP-CE mRNA (17). The hybridization signal was first detected at the internal cells of midgastrulation. The cells of C lineage, which later develop to epidermis, seemed to express AQP-CE1 mRNA. The cells expressing AQP-CE1 mRNA increased progressively until the comma stage, eventually covering the whole embryo. At the 1.5-fold stage, the expression at the tail portion started to disappear while the cells at the head portion continued to express AQP-CE1 mRNA. The mRNA expression decreased gradually from the twofold stage and disappeared before hatching. The specific role of AQP-CE1 at this relatively narrow period in C. elegans embryogenesis remains to be clarified. The functional deletion of AQP-CE1 by induction of the mutation may shed light on its physiological significance.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture, Japan, and by a grant from The Salt Science Research Foundation.
![]() |
FOOTNOTES |
---|
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: M. Kuwahara, Second Dept. of Internal Medicine, Tokyo Medical and Dental Univ., Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Received 11 May 1998; accepted in final form 12 August 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Calamita, G.,
W. R. Bishai,
G. M. Preston,
W. B. Guggino,
and
P. Agre.
Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli.
J. Biol. Chem.
270:
29063-29066,
1995
2.
Echevarria, M.,
E. E. Windhager,
and
G. Frindt.
Selectivity of the renal collecting duct water channel aquaporin-3.
J. Biol. Chem.
271:
25079-25082,
1996
3.
Fushimi, K.,
S. Sasaki,
T. Yamamoto,
M. Hayashi,
T. Furukawa,
S. Uchida,
M. Kuwahara,
K. Ishibashi,
M. Kawasaki,
I. Kihara,
and
F. Marumo.
Functional characterization and cell immunolocalization of AQP-CD water channel in kidney collecting duct.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F573-F582,
1994
4.
Ishibashi, K.,
M. Kuwahara,
Y. Gu,
Y. Kageyama,
A. Tohsaka,
F. Suzuki,
F. Marumo,
and
S. Sasaki.
Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea.
J. Biol. Chem.
272:
20782-20786,
1997
5.
Ishibashi, K.,
M. Kuwahara,
Y. Gu,
F. Marumo,
and
S. Sasaki.
Cloning and functional expression of a new aquaporin abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol.
Biochem. Biophys. Res. Commun.
244:
268-272,
1998[Medline].
6.
Ishibashi, K.,
M. Kuwahara,
Y. Kageyama,
A. Tohsaka,
F. Marumo,
and
S. Sasaki.
Cloning and functional expression of a second new aquaporin abundantly expressed in testis.
Biochem. Biophys. Res. Commun.
237:
714-718,
1997[Medline].
7.
Ishibashi, K.,
and
S. Sasaki.
The dichotomy of MIP family suggests the origin of water channels.
News Physiol. Sci.
13:
137-142,
1998.
8.
Ishibashi, K.,
S. Sasaki,
K. Fushimi,
S. Uchida,
M. Kuwahara,
H. Saito,
T. Furukawa,
K. Nakajima,
Y. Yamaguchi,
T. Gojobori,
and
F. Marumo.
Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells.
Proc. Natl. Acad. Sci. USA
91:
6269-6273,
1994[Abstract].
9.
King, L. S.,
and
P. Agre.
Pathophysiology of the aquaporin water channels.
Annu. Rev. Physiol.
58:
619-648,
1996[Medline].
10.
Kuwahara, M.,
K. Fushimi,
Y. Terada,
L. Bai,
F. Marumo,
and
S. Sasaki.
cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes.
J. Biol. Chem.
270:
10384-10387,
1995
11.
Kuwahara, M.,
Y. Gu,
K. Ishibashi,
F. Marumo,
and
S. Sasaki.
Mercury-sensitive residues and pore site in AQP3 water channel.
Biochemistry
36:
13973-13978,
1997[Medline].
12.
Le Caherec, F.,
S. Deschamps,
C. Delamarche,
I. Pellerin,
G. Bonnec,
M.-T. Guillam,
D. Thomas,
J. Gouranton,
and
J.-F. Hubert.
Molecular cloning and characterization of an insect aquaporin: functional comparison with aquaporin 1.
Eur. J. Biochem.
241:
707-715,
1996[Abstract].
13.
Ma, T.,
A. Frigeri,
H. Hasegawa,
and
A. S. Verkman.
Cloning of a water channel homolog expressed in brain meningeal cells and kidney collecting duct that functions as a stilbene-sensitive glycerol transporter.
J. Biol. Chem.
269:
21845-21849,
1994
14.
Ma, T.,
B. Yang,
W.-L. Kuo,
and
A. S. Verkman.
cDNA cloning and gene structure of a new water channel expressed exclusively in human kidney: evidence for a gene cluster of aquaporins at chromosome locus 12q13.
Genomics
35:
543-550,
1996[Medline].
15.
Maurel, C.,
J. Reizer,
J. I. Schroeder,
M. J. Chrispeels,
and
M. H. Saier, Jr.
Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes.
J. Biol. Chem.
269:
11869-11872,
1994
16.
Mitra, A. K.,
A. N. Van Hoek,
M. C. Wiener,
A. S. Verkman,
and
M. Yaeger.
The CHIP28 water channel visualized in ice by electron cryo-crystallography.
Nature Struct. Biol.
2:
726-729,
1995[Medline].
17.
NEXTDB. The nematode expression pattern database.
http://watson.genes.nig.ac.jp:8080/db/index.html.
18.
Park, J. H.,
and
M. H. Saier, Jr.
Phylogenetic characterization of the MIP family of transmembrane channel proteins.
J. Membr. Biol.
153:
171-180,
1996[Medline].
19.
Wall, D. A.,
and
S. Patel.
Isolation of plasma membrane complexes from Xenopus oocytes.
J. Membr. Biol.
107:
189-201,
1989[Medline].
20.
Waltz, T.,
B. L. Smith,
P. Agre,
and
A. Engel.
The three-dimensional structure of human erythrocyte aquaporin CHIP.
EMBO J.
13:
2985-2993,
1994[Abstract].
21.
Weig, A.,
C. Deswarte,
and
M. J. Chrispeels.
The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group.
Plant Physiol.
114:
1347-1357,
1997