(Received for publication, October 2, 1995)
From the
Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel in the kidney collecting duct. AQP-2 is selectively permeable to water molecule and is translocated between the apical membrane and subapical endosomes in response to vasopressin. To investigate the localization and structure of the aqueous pathway of the AQP-2 water channel, a series of site-directed mutants was constructed and functionally analyzed. Insertion of N-glycosylation reporter sequence into each hydrophilic loop (HL) indicated that AQP-2 has a six-membrane spanning topology and that insertional mutations in HL-2 or HL-5 do not alter water channel function. Mercury-sensitive site of AQP-2 is located near the second asparagine-proline-alanine (NPA) domain at cysteine 181, but not near the first NPA domain. Replacement of HL-3 or HL-4 with the corresponding part of Escherichia coli glycerol facilitator abolished water channel function without changing plasma membrane expression of the channel protein. Introduction of cysteine residues in His-122, Asn-123, Gly-154, Asp-155, or Asn-156 induced partial mercury sensitivity, and point mutations in asparagine 123 significantly altered water permeability. Our results implicate that the structure of AQP-2 is different from models previously proposed for AQP-1 and that HL-3 and HL-4 are closely located to the aqueous pathway.
Aquaporin-2 (AQP-2, ()previously reported as WCH-CD
or AQP-CD) is a water channel in the apical membrane of the kidney
collecting duct(1) . Water permeability of this nephron segment
is regulated by vasopressin through the membrane shuttle
mechanism(2, 3, 4) , in a mechanism by which
AQP-2 is translocated between the apical membrane and endosomes under
vasopressin regulation(5, 6, 7) .
Complementary DNAs for rat and human AQP-2 have been
isolated(8, 9) , and the primary structure of AQP-2
has been identified. AQP-2 is a very hydrophobic membrane-integral
protein of a molecular mass of
29 kDa. It is a member of the MIP
protein family (10) and is homologous to aquaporin-1 (AQP-1,
previously reported as CHIP28)(11, 12, 13) .
Functional expression of AQP-2 showed that it is highly permeable to
water molecule but not to urea, glycerol, and ions and that its water
permeability is mercury-sensitive and
temperature-insensitive(8) . Furthermore, it has been shown
that mutations in AQP-2 gene are responsible for deficient vasopressin
antidiuresis in some patients with nephrogenic diabetes
insipidus(14, 15) .
Despite accumulated knowledge
of AQP-2 physiology implicating functional importance of AQP-2 in urine
concentration and homeostasis of body fluid, the molecular structural
basis of AQP-2 is not well known. The localization and higher order
structure of the aqueous pathway of AQP-2 have to be elucidated to
account for its selective permeability to water molecule and explain
the mutation-related channel malfunction. The molecular structure of
AQP-1, the first identified water channel, has been studied and
partially resolved. It was shown that AQP-1 exists in plasma membrane
with tetramer formation(16, 17) but that each monomer
is functionally independent, thus leading to the assumption that single
aqueous pore spans each monomer(18, 19) . Regarding
the structure of the aqueous pore in functionally active AQP-1 monomer,
three structural models have been proposed(20) : the hourglass
model(21) , the -helical model(22) , and the
-barrel model(23) . According to the hourglass model, the
aqueous pathway is formed by two domains with an NPA box, which is an
asparagine-proline-alanine sequence highly conserved among the MIP
family members. In the
-helical model, the aqueous pathway is
located between transmembrane segments with
-helical conformation.
In the
-barrel model, the aqueous pore is formed with
16
antiparallel
-sheets analogous to a porin channel of bacteria
species(24) . The validity of these models has not been
sufficiently examined. Moreover, there have been no investigations
regarding the molecular structure of aquaporins other than AQP-1. In
addition to being significant for understanding the general structure
of channels, elucidation of the structure of aquaporins will provide
insights for the development of an AQP-2 inhibitor, which is
potentially of remarkable use as a water diuretic.
This study examined membrane topology, mercury-sensitive sites, and functional roles of the two of the NPA-containing domains and other hydrophilic loops of AQP-2. We found significant participation of hydrophilic loops other than NPA-containing domains in the formation of the aqueous pathway of AQP-2. Based on our observations, a new structural model for AQP-2 is proposed.
Figure 1: A schematic for structure of AQP-2. Proposed membrane topology of AQP-2 and designs for mutagenesis experiments are shown. AQP-2 is presumably composed of six transmembrane segments, amino (N) and carboxyl (C) termini located cytoplasmically, and five connecting hydrophilic loops, numbered 1-5. Conservative NPA sequences are illustrated as open boxes. Potential N-glycosylation site of wild-type AQP-2 is marked by an asterisk. Alternative N-glycosylation signals were introduced into each hydrophilic loop at positions indicated as 36NTS, 65NTS, 154NTS, or 194NTS. Potential mercury-sensitive sites of Cys-181 and Ala-65 are indicated by open circles. Recombinations of HL-3 or HL-4 were made at positions indicated by hatched areas.
The effects of mercury reagents were examined by incubating
oocytes in Barth's buffer containing 1 mM HgCl for 10 min prior to P
measurements.
Figure 2:
Native and alternative N-glycosylation of AQP-2 expressed in rat kidney and oocytes
injected with wild-type or mutant AQP-2 cRNA. Immunoblot analysis with
antibody against COOH-terminal synthetic peptide is shown. A,
10 µg of rat kidney membrane fraction protein and membrane
fractions from oocytes injected with wild-type AQP-2 cRNA pretreated
without(-) or with (+) N-glycosidase F. B,
immunoblot analysis of total membrane fractions from oocytes expressing
NTS insertional mutants is shown. Total membranes from one oocyte for
wild type (lane 1), N124D (lane 2), 36NTS (lane
3), 65NTS (lane 4), 154NTS (lane 5), and 194NTS (lane 6) not treated with N-glycosidase F are
blotted. 36NTS (lane 7) and 194NTS (lane 8) were
digested with N-glycosidase F and blotted. Positions for
molecular mass markers are shown. Arrow head, 29-kDa
AQP-2 core protein.
Figure 3:
Osmotic water permeability of oocytes
expressing N-glycosylation mutants of AQP-2. Summary of a
series of osmotic water permeability (P)
measurements (n = 20-30) are shown. Hatched
bar, mean P
of oocytes injected with
wild-type and mutant cRNA; open bar, P
of oocytes after incubation with Barth's buffer
containing 1 mM HgCl
for 10 min. Data are shown as
means and S.E.
As the mercury-sensitive site is expected to be localized close to the aqueous pore(21) , inhibition by mercury agent was examined for a series of cysteine mutants. When cysteine at 181 was replaced, mercury inhibition disappeared in contrast to other substitutions of C75S, C79S, and C144S; replacements of cysteine 181 with larger residues inhibited water permeability, suggesting cysteine 181 is localized near the aqueous pathway (Fig. 4). Substitution by cysteine of alanine at 65, an alternative mercury-sensitive site proposed by the hourglass model, in mercury-resistant mutant C181A did not induce mercury sensitivity. Localization of mercury-sensitive site in the first NPA domain, something which strongly supports the hourglass model for AQP-1, was not observed in AQP-2, implicating the difference in the structures of the aqueous pathways of AQP-1 and AQP-2. Observations from NTS insertion and cysteine substitution taken together indicated that cooperative participations of HL-2 and HL-5 in the formation of the aqueous pathway of AQP-2 is not as critical as proposed in the hourglass model for AQP-1.
Figure 4:
Osmotic water permeability and mercury
sensitivity of oocytes expressing AQP-2 with mutations in hydrophilic
loop-3 and loop-4. Osmotic water permeability (P) of oocytes injected with water or 2
ng of indicated cRNAs is shown on the left with dotted
bars. Mercury sensitivity indicated as percent inhibition by
incubation with 1 mM HgCl
for 10 min is shown on
the right with hatched bars. Means and S.E. of
20-40 experiments are presented. A65C/C181A, double mutations of
A65C and C181A; HL3-GlpF: recombinant of HL-3 with GlpF; HL4-GlpF,
recombinant of HL-4 with GlpF; HL4-AQP1, recombinant of HL-4 with
AQP-1; HL4-MIP, recombinant of HL-4 with MIP; WT, wild type; ND, not determined.
Figure 5: Immunoblot analysis of total membrane and plasma membrane fractions from oocytes expressing AQP-2 with recombination of hydrophilic loop-3 (HL3) and loop-4 (HL4) with GlpF. Total membrane fractions from 1 oocyte (left) and plasma membrane fractions from 20 oocytes (right) were separated by SDS-PAGE and blotted. Positions for molecular mass markers are shown on the left. Arrow head, 29-kDa core protein of AQP-2.
A series of
single-residue substitutions in HL-3 and HL-4 was performed to
investigate the interaction of these loops with the aqueous pathway.
Replacement of native residues to cysteine has successfully been used
to determine the localization of the aqueous pathway of aquaporins (21) . The rationale is that there is a high probability that
cysteine residues near the aqueous pathway interact with imposed
mercury agents and inhibit water channel function. When a cysteine
residue was introduced into amino acid 122 or 123 of mercury-resistant
mutant of AQP-2, C181A, up to 40% inhibition by low concentration of
HgCl was observed, which was significantly higher than that
for C181A (p < 0.001, n = 20). The mercury
inhibition, although it is not complete, implicated that Asn-123 is
likely to be located near the aqueous pathway. Subsequently, a residue
Asn-123 was replaced with a series of amino acids to examine the
interaction of the lateral moiety with the aqueous pathway. Osmotic
water permeability was decreased roughly in accordance with the size of
the lateral moiety (Fig. 4). This may be interpreted as the
localization of the constriction of the aqueous pathway at Asn-123 and
the occlusion by the larger lateral moiety. Plasma membrane expressions
of the series of Asn-123 mutants were comparable to those of the wild
type as assessed by immunoblot (Fig. 6) and radioimmunoassay (60
± 10% for N123A, 65 ± 12% for N123Q, 65 ± 12% for
N123D, 65 ± 12% for N123W, relative to the wild type, n = 3-5), showing substantial expression of N123
mutants in the plasma membrane. A similar analysis was done for HL-4.
Cysteines in 154, 155, and 156 exhibited high mercury sensitivity, with
the highest at Asp-155 (p < 0.001 versus C181A, n = 20), implicating close localization of Asp-155 to
presumed aqueous pathway.
Figure 6: Immunoblot analysis of total membrane and plasma membrane fractions from oocytes expressing Asn-123 mutants. Total membrane fractions from 1 oocyte (T) and plasma membrane fractions from 20 oocytes (P) were separated by SDS-PAGE and blotted. Positions for molecular mass markers are shown on the left. WT, wild type.
In this study, we have examined the structure of the aqueous pathway of rat AQP-2 and found that the contribution of HL-3 and HL-4 is significant in the formation of the aqueous pathway. It was suggested that the structure of the aqueous pore is somehow different from that of AQP-1 proposed previously(21, 22, 23) . A six-transmembrane topology and mercury-sensitive positions in AQP-2 are identical to AQP-1 hourglass model(21) . However, mutations near NPA boxes in HL-2 and HL-5 did not significantly alter water channel function, and cysteine residues inserted near the first NPA box did not induce the alternative mercury-sensitive site. Alternatively, based on the findings that the replacement of HL-3 and HL-4 with hydrophobic residues decreased water permeability without affecting plasma membrane expression and that mercury sensitivity was found in these domains, it was suggested that HL-3 and HL-4 are located near the aqueous pore.
Membrane topology and mercury-sensitive cysteine residues have first
been determined experimentally in this study, and it was shown that
AQP-2 is similar to AQP-1 proposed as the hourglass model in its
topological presentation. Membrane topology was partially determined
from the presence of N-glycosylation, which indicates
extracellular localization of the site. N-Glycosylation site
insertion has been successfully used to map membrane topology of the
cystic fibrosis transmembrane regulator (25) and the glutamate
receptor (32) . The N-glycosylation insertion was used
in the present study because the glycosylation is natural and the
consensus glycosylation sequence minimally perturbs the native sequence
and structure(25) . Immunoblot results indicated the
extracellular localization of HL-1, HL-3, and HL-5, implicating that
the -helical model (22) and the
-barrel model (23) are not adequate for the structure of AQP-2. Sidedness of
both termini, HL-2 and HL-4, cannot be determined because the
extracellular NTS motif is not always glycosylated. However, when the
evidence of phosphorylation of the carboxyl terminus of AQP-2 is also
taken into account(30) , it may be reasonable to conclude that
AQP-2 has a six-transmembrane topology identical to that of AQP-1
proposed in the hourglass model(21, 26) .
Inhibition of osmotic water permeability by low concentration of
mercury chemicals is characteristic of protein water channel, and
mercury is believed to interact with cysteine residues located near the
aqueous pore, perturbing the structure and function of the aqueous pore (21) . Therefore, identification of the mercury-sensitive
cysteine residue is critical for locating the aqueous pore. The results
showed that the mercury-sensitive cysteine of AQP-2 is Cys-181, which
corresponds to Cys-189 of AQP-1. In addition, molecular size-dependent
impairment of water permeability was observed in Cys-181 replacement,
which was similar to observations in AQP-1. When Cys-181 was replaced
with tryptophan, P had the lowest value. Because
the plasma membrane expression of C181 mutants was similar as assessed
by antibody binding and membrane fractionation (data not shown), it is
likely, as proposed in the AQP-1 experiments(18) , that
replacement of Cys-181 with larger residues interfered with the aqueous
pathway. These data suggested that the aqeuous pore of AQP-2 may be
closely located to Cys-181.
Although identical topology and the mercury-sensitive site of AQP-2 and AQP-1 reasonably implicated the structural similarity of the two channels, our observation from mutational analysis indicated that the structure of the aqueous pathway of AQP-2 is somehow different from that of AQP-1. Although the contribution of HL-2 and HL-5 to the formation of the aqueous pathway has been strongly postulated in the hourglass model for AQP-1(21) , our results were not compatible with the previous observations. First, introduction of cysteine to Ala-65, which corresponds to Ala-73 of AQP-1, did not induce mercury sensitivity. Symmetrical localization of mercury-sensitive sites in Cys-189 and Ala-73 were one of the bases for the hourglass model for AQP-1. Second, insertion of a few amino acid residues into HL-2 and HL-5 of AQP-2 did not affect water channel function. Inhibition of water permeability by the insertion of the BamHI sequence into the corresponding sites of HL-2 or HL-5 supported the hourglass model of AQP-1(26) . In the hourglass model, it was claimed that HL-2 and HL-5, being folded into the lipid bilayer, partially form the aqueous pore(21) . However, a glycosylation reporter sequence inserted into HL-5 was properly glycosylated with minimal effects on water permeability, showing extracellular localization of this part. Our results may indicate that the hourglass model is not adequate for describing the aqueous pore of AQP-2.
Functional analysis of
replacement, mercury sensitivity, and point mutations in HL-3 and HL-4
implicated that these loops participate in the formation of the aqueous
pathway of AQP-2. HL-3 and HL-4 are relatively hydrophilic compared to
other segments of AQP-2. Hydropathy analysis of other aquaporins
including AQP-1, AQP-3, MIP, and -TIP showed that hydrophilicity
of these domains is a common feature of water
channels(33, 34) . Thus, it is reasonable to speculate
that HL-3 and HL-4 contribute to the structure of the aqueous pore and
to selective water permeability. Disappearance of water permeability by
the replacement of HL-4 with GlpF but not with AQP-1 or MIP implicated
that hydrophilicity of this domain may be critical for water
permeability. Furthermore, the findings that mercury sensitivity was
maximum at positions Asn-123 and Asp-155 and that substitutions of
Asn-123 with larger residues decreased P
can be
interpreted that Asn-123 and Asp-155 are the closest to the presumed
constriction of the aqueous pore of an expected size of
2 Å (21, 22, 23) . However, our observations may
raise several questions, which must be resolved before any definitive
conclusions can be made. First, although mercury inhibition found in
HL-3 and HL-4 was significant compared to C181A, it was lower than that
for wild type. The lower mercury sensitivity can be explained by the
possibility that HL-3 and HL-4 are located further to the aqueous
pathway than Cys-181. Second, insertion of NTS after the residue 154,
which should be the critical site for water channel function according
to our data, did not alter P
. This may be because
hydrophilicity of inserted residues minimally disturbed the pore
structure. Further structural analyses will be required to interpret
our current observations and those of the previous studies and to
describe a more precise structure of the aqueous pore.
Care must be taken for the interpretation of mutational analysis of aquaporins because it has been well known that mutations in aquaporins sometimes disturb synthesis, assembling, and plasma membrane expression of mutant proteins in oocytes(19, 21, 35) . Thus, functions of mutated channel have to be normalized with the amount of protein in the plasma membrane. Therefore, we undertook immunological analysis of synthesized mutants to ensure that mutants were readily synthesized and expressed in the plasma membrane. Plasma membrane expression of mutated channel was successfully resolved by two methods. For this purpose, we did not use immunohistochemical staining of oocyte membranes, which is less quantitative compared to the two methods we used. All mutations in HL-3 and HL-4 examined here did not significantly affect plasma membrane expression, confirming that the mutations only minimally perturb the channel structure.
On the basis
of our results, we propose a structure model for AQP-2 water channel (Fig. 7). Between six transmembrane -helices, a central
channel may be formed, the diameter of which would be larger than the
expected size for selective aqueous pore. Constrictions with a pore
size of
2 Å that determine selective water permeability may
be assembled in the association of HL-3 and HL-5 in the extracellular
side and in the association of HL-2 and HL-4 in the cytoplasmic side.
These hydrophilic loops may be partially folded into the large pore,
forming constrictions and hydrophilic apparatus that exclude charged
ions and large molecules. Since N-glycosylation of HL-3 or
HL-5 did not influence pore structure, it is likely that these domains
are not folded deep into lipid bilayer as described in the hourglass
model. The roles of the NPA boxes, which are strictly conserved among
the MIP family, are not clear from our study. It is speculated that the
NPA boxes are critical to correctly assemble three-dimensional
structures of aquaporins because AQP-2 proteins with mutations near NPA
boxes were suggested to be folded improperly(35) .
Figure 7: Model of proposed structure of AQP-2 water channel. A schematic model of the structure and the aqueous pathway of AQP-2. The aqueous pathway is assembled with six transmembrane segments (hatched box with numbers I-VI), hydrophilic loop-3 (HL3), loop-4 (HL4), and two of the NPA-containing domains (NPA1, NPA2). A pore structure and constrictions that may determine selective water permeability are assembled with HL-3 and the second NPA domain (NPA2) in the extracellular side and with HL-4 and the first NPA domain (NPA1) in the cytoplasmic side.