©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mercurial Insensitive Water Channel (AQP-4) Forms Orthogonal Arrays in Stably Transfected Chinese Hamster Ovary Cells (*)

(Received for publication, December 8, 1995; and in revised form, January 8, 1996)

Baoxue Yang Dennis Brown (1) A. S. Verkman (§)

From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521 and Department of Pathology and Renal Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mercurial insensitive water channel (MIWC, AQP-4) is a water-selective transporter expressed at the basolateral plasma membrane of principal cells in kidney collecting duct, airway epithelium, and gastric parietal cells, as well as in astrocytes and skeletal muscle plasmalemma. Because these sites correspond to membranes where orthogonal arrays of particles (OAPs) have been observed by freeze-fracture electron microscopy, we tested the hypothesis that MIWC forms OAPs. Chinese hamster ovary cells were stably transfected with the coding sequence of rat MIWC under a cytomegalovirus promoter. Immunostaining of clonal cell populations showed MIWC expression at the plasma membrane. A single band at 31 kDa was detected on immunoblot. Cell fractionation by sucrose gradient centrifugation indicated strong MIWC expression in plasma membrane fractions with lesser expression in Golgi. Functional analysis by stopped-flow light scattering showed high mercurial insensitive water permeability in plasma membrane vesicles. Freeze-fracture electron microscopy showed distinct OAPs on the plasma membrane P-face of MIWC-expressing cells with morphology indistinguishable from that in basolateral membrane of kidney collecting duct; the E-face showed corresponding linear grooves (spacing, 8 nm) in transfected cells and collecting duct. OAPs were not observed in control (empty vector-transfected) cells or CHIP28 (AQP1)-transfected cells in which disorganized intramembrane particle aggregates were found. These results provide direct evidence that a molecular water channel can spontaneously assemble in regular arrays.


INTRODUCTION

A family of water transporting proteins (``water channels'' or ``aquaporins'') has been identified recently, which are small (30 kDa) integral membrane proteins with homology to the major intrinsic protein (MIP) (^1)of lens fiber (for review, see (1, 2, 3) ). Many years prior to the molecular identification of water channels, characteristic patterns of intramembrane particles (IMPs) observed by freeze-fracture electron microscopy were proposed to represent a morphological signature of water-transporting units. In amphibian skin, regular square arrays of IMPs, called orthogonal arrays of particles (OAPs), were found on the plasma membrane of water-permeable epithelial cells after stimulation by vasopressin or isoproterenol(4) . In toad bladder, vasopressin-induced IMP aggregates with a similar morphology are induced by vasopressin(5, 6) ; in kidney collecting duct, vasopressin induces the appearance of less organized clusters of IMPs on the apical membrane of principal cells(7, 8) . However, the basolateral plasma membrane of the same collecting duct cell type contains large numbers of OAPs that appear similar to the vasopressin-induced OAPs in amphibian skin(9, 10) .

A series of morphological studies have indicated that OAPs are located in the plasma membranes of many different cell types in mammals: the basolateral membrane of principal cells in kidney collecting duct(10, 11) , epithelial cells in trachea(12) , intestine(13) , ciliary body (14) , and stomach(15) , brain astrocytes(16, 17) , skeletal muscle(18, 19) , and lens fiber. The OAPs in lens fibers were shown to contain MIP26, a protein expressed only in lens fiber that is homologous to all members of the aquaporin family but which has only limited water channel function(20, 21, 22, 23) . The identity and function of the OAPs in other tissues have remained enigmatic since their first discovery in astrocytes over two decades ago(17) .

We recently proposed, based on tissue distribution studies and homology to MIP, that the mercurial insensitive water channel (MIWC) may be the OAP protein(24) . MIWC was cloned from rat lung(25) , and subsequently an isoform with an extended NH(2) terminus was cloned from rat brain (AQP-4, (26) ). MIWC is unique among the five cloned mammalian water channels in that its water transport function is not inhibited by mercurials because of the absence of a cysteine residue near its putative aqueous pore(27) . Rat MIWC is a non-glycosylated, 301-amino acid protein that spans the membrane six times with cytosolic NH(2) and COOH termini(28) . Immunolocalization showed rat MIWC protein expression at the basolateral plasma membrane of kidney collecting duct, astrocytes in brain and spinal cord, and epithelial cells in stomach, trachea, bronchi, intestine, ciliary body, and various exocrine glands(22, 29) . A human homolog of MIWC is found as a single copy gene at chromosome locus 18q22 and encodes distinct protein isoforms utilizing distinct transcripts generated by alternative splicing(30) . The immunolocalization data, taken together with functional studies in kidney (31) and airways(32) , suggested a potential role for MIWC in the urinary concentrating mechanism, cerebrospinal fluid reabsorption, airway hydration, and glandular secretions.

The purpose of this study was to test the hypothesis that MIWC is able to form orthogonal arrays in a heterologous transfected cell model. CHO cells were stably transfected with rat MIWC utilizing procedures applied previously to generate cells that expressed functional and correctly targeted water channels CHIP28 (AQP-1) and AQP-2(33, 34) . The transfected cells expressed MIWC as a functional 31-kDa protein at the cell plasma membrane. Freeze-fracture electron microscopy showed prominent OAPs in MIWC-expressing cells that were morphologically indistinguishable from OAPs present in the basolateral membrane of kidney collecting duct.


EXPERIMENTAL PROCEDURES

Plasmid Constructions

The 903-base pair coding sequence of rat MIWC was polymerase chain reaction-amplified using rat lung MIWC cDNA as template and primers (sense, 5`-GCTGATCATGGTGGCTTTCAAAGGC-3`; antisense, 5`-GCTCTAGATACAGAAGATAATACCTC-3`) with engineered BclI (5`) and XbaI (3`) restriction sites. The BclI and XbaI fragment was ligated into mammalian expression vector pcDNA3 (Invitrogen) at BamHI and XbaI restriction sites and propagated in Escherichia coli. The construct was confirmed by sequence analysis.

Cell Culture and Transfection

CHO-K1 (wild type) cells (UCSF Cell Culture Facility) were grown in Ham's nutrient mix supplemented with 10% fetal calf serum at 37 °C in 5% CO(2). Cells were plated at a density of 5 times 10^5/60-mm diameter dish 12 h before transfection. Cells were washed with Opti-MEM1 media (Life Technologies, Inc.) and then incubated for 12 h at 37 °C with 2 ml of medium containing 20 µg of Lipofectin (Life Technologies, Inc.) and 10 µg of recombinant plasmid (or empty plasmid without insert). Cells were then washed and incubated for 24 h in 4 ml of Ham's nutrient mix containing 10% fetal calf serum. Cells were trypsinized and plated on 100-mm diameter dishes, and Geneticin (G418, Life Technologies, Inc.) was added (500 µg/ml) for selection. At 10-14 days, G418-resistant cell clones were isolated and transferred to separate culture dishes for expansion and analysis.

Subcellular Fractionation

Fractionation was performed by a modification of reported methods(33, 35) . Cells from ten 25-cm diameter plastic dishes were grown to confluence (1-3 times 10^8 cells/dish), released by agitation in PBS containing 8 mM EGTA, and washed twice (100 times g, 10 min) at 4 °C in homogenizing buffer (HB, 250 mM sucrose, 10 mM Tris-HCl, pH 7.4). The pellet was resuspended in HB containing antipain (1 µg/ml), pepstatin (1 µg/ml), and benzamidine (15 µg/ml) and homogenized by 20 strokes of a glass Dounce homogenizer. The homogenate was centrifuged at 500 times g for 10 min at 4 °C and adjusted to 1.4 M sucrose, 10 mM Tris-HCl, 0.2 mM EDTA (pH 7.4). A discontinuous sucrose gradient (2 M sucrose (1 ml), 1.6 M (2 ml), 1.4 M (4 ml, containing homogenate), 1.2 M (4 ml), 0.8 M (1 ml)) was centrifuged for 2.5 h at 25,000 rpm in an SW 27 rotor, and 1-ml fractions were collected. Protein concentration was determined by the Bradford method. Enzyme activities for alpha-glucosidase and alkaline phosphodiesterase I were determined as reported previously(33) .

Water Transport Assay

Osmotic water permeability (P(f)) was measured by stopped-flow light scattering (36) in vesicle fractions suspended at 0.5 mg of protein/ml in 50 mM mannitol, 5 mM sodium phosphate, pH 7.4. Vesicles were subjected to a 100 mM inwardly directed sucrose gradient, and the time course of scattered light intensity at 530 nm was measured.

Immunoblot Analysis

Cells (homogenized in 200 mM sucrose, 10 mM Tris-HCl, pH 7.4, containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 4 µg/ml antipain) or membrane fractions were heated to 70 °C for 10 min in SDS loading buffer, resolved on a 12% polyacrylamide gel, and electrotransferred to a polyvinylidene difluoride membrane. After blocking with 2% bovine serum albumin in PBS for 1 h, a 1:1000 dilution of control or immune serum (rabbit polyclonal antiserum prepared as described in (29) ) was added for 2 h. The membrane was washed in PBS, incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:1000, Life Technologies, Inc.), and developed with 5-bromo-4-chloro-3-indolylphosphate (16 µg/ml) and nitroblue tetrazolium (0.33 µg/ml).

Immunofluorescence

Cells grown on glass coverslips were fixed with 4% formaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The polyclonal anti-MIWC antibody (or preimmune serum control) was incubated in PBS containing 3% bovine serum albumin and 0.2% Triton X-100 for 1 h at room temperature and then washed four times with PBS containing 1% Triton X-100. Cells were then incubated with the secondary rhodamine-conjugated goat anti-rabbit antibody (1:500 dilution, Life Technologies, Inc.) for 1 h and washed four times in PBS containing 1% Triton X-100. Slides were viewed in a Leitz-Technical Instruments epifluorescence confocal microscope with cooled CCD camera detector. Confocal images were obtained with a times 60 objective (Nikon, oil immersion, numerical aperture 1.4).

Freeze-Fracture Electron Microscopy

Cells grown on plastic 6-well plates were fixed for 2 h in 2% glutaraldehyde, rinsed, and stored in PBS. Monolayers were cryoprotected in 30% glycerol for at least 1 h before scraping. A small aliquot of the cell suspension was placed on a copper specimen holder and frozen by immersion in Freon 22 cooled with liquid nitrogen. Specimens were fractured and coated with platinum-carbon in a Cressington freeze-fracture device as described previously(37) . Replicas were cleaned in sodium hypochlorite bleach and chloroform/methanol (2:1) before being examined and photographed in a Philips CM10 electron microscope.


RESULTS AND DISCUSSION

Clonal cell populations of stably transfected cells expressing MIWC had identical growth characteristics and morphology to control cells transfected with empty vector and selected identically. Cell clones were first analyzed for MIWC expression, membrane localization, and function. Immunofluorescence with a rabbit polyclonal anti-MIWC antibody showed MIWC distribution in a membrane pattern with staining often observed in a perinuclear location probably corresponding to Golgi (Fig. 1A). Staining of control (vector-transfected) cells with immune serum was minimal (Fig. 1B), as was staining of MIWC-transfected cells with preimmune serum (not shown). High magnification confocal microscopy (Fig. 1C) indicated a plasma membrane distribution for MIWC.


Figure 1: Immunofluorescence of transfected CHO cells with anti-MIWC antibody. MIWC-transfected (A) and control (empty vector-transfected, B) cells were examined by indirect immunofluorescence as described under ``Experimental Procedures.'' Cells were viewed by wide-field fluorescence microscopy with times 20 dry objective. C, confocal micrograph of MIWC-transfected cells with times 60 oil immersion objective.



Subcellular fractionation was carried out to confirm the tissue distribution of immunoreactive MIWC and to isolate vesicles suitable for functional analysis. Fractionation by sucrose density gradient centrifugation yielded fractions enriched in endoplasmic reticulum, Golgi membranes, and plasma membranes. The identification of fractions was carried out by marker enzyme assays as described previously(33) . Immunoblot analysis in Fig. 2A (left) showed prominent expression of MIWC in the cell homogenate and the plasma membrane fraction; there was in some blots a weak band detectable in the Golgi fraction. MIWC was seen as a single band on immunoblot without glycosylation, similar to immunoblot findings in native cell membranes(24) . No distinct bands were detected in immunoblots of membranes from control cells probed with immune serum (Fig. 2A, right) or membranes from MIWC-expressing cells probed with preimmune serum (not shown).


Figure 2: Immunoblot and functional analysis of subcellular fractions. A, immunoblot performed on cell homogenates and isolated vesicles fractions as described under ``Experimental Procedures.'' Each lane contains 3 µg (homogenate) and 1 µg (vesicle fractions) of total protein. B, stopped-flow light scattering measurement of osmotic water permeability. Vesicles were subjected to a 100 mM inwardly directed sucrose gradient at 10 °C, and 90° scattered light intensity was measured at 530 nm. Average vesicles diameters were measured by quasi-elastic light scattering to be 225 nm (endoplasmic reticulum (ER)), 157 nm (Golgi), and 160 nm (plasma membranes). See text for P.



Functional analysis of water permeability was conducted on the isolated membrane fractions. Fig. 2B shows stopped-flow light scattering measurements of osmotic water permeability in which vesicle suspensions were subjected to a 100 mM inwardly directed gradient of sucrose as described under ``Experimental Procedures.'' The gradient caused vesicle shrinkage, which produced a change in scattered light intensity. The time course of light scattering was similar in endoplasmic reticulum from the control and MIWC-expressing cells. Shrinkage was slightly faster in Golgi vesicles but much faster in plasma membrane vesicles from MIWC-expressing cells than from control cells. Calculated osmotic water permeability (P(f)) for the plasma membrane vesicles was 0.018 cm/s at 10 °C. There was no effect of 0.3 mM HgCl(2) on P(f) (not shown), a concentration of HgCl(2) that strongly inhibited water permeability mediated by CHIP28(36) . Stopped-flow light scattering studies performed with urea and glycerol as solute (100 mM inwardly directed gradients) showed that MIWC is selective for passage of water. These results are consistent with functional measurements in Xenopus oocytes expressing MIWC(25) .

Freeze-fracture electron microscopy of cells transfected with MIWC cDNA revealed aggregates of intramembrane particles scattered on the plasma membrane P-face. In each membrane fragment from 11 separate cells examined from two different cultures, the particles comprising these aggregates were arranged into orthogonal arrays (Fig. 3a) that closely resemble the OAPs seen in other cell types, including the basolateral plasma membrane of collecting duct principal cells (Fig. 3b). In addition to large OAPs, the cell membrane in Fig. 3a also contained many large IMPs that appeared more numerous than those present in control cells. These large IMPs could represent MIWC proteins that are not assembled into characteristic OAPs. On the plasma membrane E-face of MIWC-expressing cells, corresponding linear arrays of grooves were frequently observed (Fig. 3c). The precise appearance of the E-face grooves depended on the direction of platinum shadowing relative to the groove array. In this case, the imprint appeared as a series of linear grooves (arrows), each formed by a series of small closely apposed depressions in the membrane leaflet. These arrays of grooves were similar to those seen on the basolateral membrane E-face of collecting duct principal cells (Fig. 3d). In some areas, parallel arrays of grooves are seen (arrows) that were indistinguishable from the arrays of grooves in the MIWC-transfected CHO cells. These organized arrays were never seen on control (vector-transfected) cells (Fig. 3e) nor were they found in cells transfected with CHIP28 (Fig. 3f). Many large IMPs, previously shown to correspond to CHIP28 tetramers(37, 38) , are present on the plasma membrane. While they are sometimes concentrated into dense clusters of IMPs, no organized geometric arrangement of the IMPs was seen. OAPs were also never seen in freeze-fracture micrographs of LLC-PK1 and Madin-Darby canine kidney cells as well as CHO cells transfected with the AQP-2 protein (not shown).


Figure 3: Freeze-fracture electron micrographs of CHO cell plasma membranes and collecting duct principal cell basolateral plasma membranes. a, large OAPs on the P-face of a MIWC-expressing CHO cell. The particles are arranged in an orthogonal, geometric pattern characteristic of OAPs from other tissues. b, OAPs in the basolateral membrane P-face of a collecting duct principal cell. c, plasma membrane E-face of a MIWC-transfected CHO cell showing an array of parallel, linear grooves that represent the imprints of P-face OAPs shown in a. d, E-face of the basolateral plasma membrane of a collecting duct principal cell showing the appearance of imprints left in this membrane leaflet by the P-face OAP arrays. e, plasma membrane P-face from a non-transfected CHO cell showing small intramembrane particles, randomly distributed on the membrane. No large IMP clusters or arrays were detected. f, plasma membrane P-face from CHO cells expressing CHIP28 (AQP-1). Bars: 70 nm (a, b, e, f) and 50 nm (c, d).



In Fig. 3, c and d, the center-to-center spacing of the E-face grooves was 8 nm, as was the center-to-center spacing of the indentations along the wall of each groove. On the P-face (Fig. 3, a and b), each large square IMP was 16 times 16 nm in size and in many cases appeared to be composed of four subunits (center-to-center spacing, 8 nm) when examined at higher magnification. Thus, the grooves probably correspond to impressions produced by the four subunits of the larger IMP. Based on previous analysis of CHIP28 IMP size(37) , each 8-nm ``subunit'' might consist of a MIWC tetramer.

Taken together with previous data(20, 21, 37, 38, 39) , the results here indicate that MIWC and MIP spontaneously assemble into OAPs in a heterologous expression system and/or in proteoliposomes, whereas CHIP28 and AQP-2 generally do not. Highly purified CHIP28 does assembly into two-dimensional crystalline arrays when reconstituted into liposomes at very high protein-to-lipid ratios under conditions of slow detergent removal(40, 41) ; however, the CHIP28 orientation is very different from that in real membranes in that adjacent CHIP28 molecules in the crystal are oriented in opposite directions.

The physical interactions that drive the formation of OAPs are not known. Because of the close proximity between adjacent protein units, protein-protein interactions may provide the driving force for OAP formation such as interhelix dipole-dipole coupling and attraction between polar residues. We carried out hydropathy(22) , helical wheel (42) , and helix dipole (43) calculations for MIWC and MIP (proteins that form OAPs) and CHIP28 and AQP-2 (proteins that do not). The sequences for each putative helical domain were aligned, hydropathy values were compared before and with arbitrary helix rotation, and dipole moments were computed. However, no significant difference was found that distinguished MIWC and MIP from their homologs. The driving force for OAP assembly may involve protein and/or lipid interactions that were not considered, or the free energy for individual helix-helix interactions may be too small to detect computationally.

Is there functional significance to the assembly of water channel proteins in OAPs? Biophysical analysis has indicated that the single channel water permeability of CHIP28 is low (<10 cm^3/s; (36) ) so that water channel density must be very high (>1000 channelsbulletµm^2) to increase water permeability significantly above channel-independent water permeability through lipids(1) . For CHIP28, each monomer functions as an independent water channel(44, 45) , giving no advantage to specific supermolecular assemblies. In contrast, MIWC was found recently to display subunit-subunit interactions, whereby an adjacent mutant subunit affected the function of wild-type MIWC(27) . Such interactions, which might occur in adjacent units in OAPs, might enhance MIWC water permeability. Theoretically, it is possible that parallel movement of water through closely spaced channels might augment net osmosis by a bulk flow mechanism(46) ; experimental verification of this hypothesis will be needed utilizing membrane systems having different MIWC assemblies. Another potential advantage for regular OAPs is to maximize water channel surface density while minimizing protein-protein contact.

Our data suggest that OAPs in plasma membranes of many different cell types are composed of the water-selective transporting protein MIWC. Interestingly, modification of the membrane content and/or the morphological appearance of these OAPs has been reported for various physiological stimuli and pathological states, including stimulation of acid secretion by pentagastrin in parietal cells(47) , brain ischemia (48, 49) , aluminum-associated epilepsy in astrocytes(50) , and muscular dystrophy(51) . Further studies are required to determine the significance of these observations.


FOOTNOTES

*
This work was supported by Grants DK35124, HL42368, DK36451, and HL51854 from the National Institutes of Health and RDP Grant R613 from the National Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 1246 Health Sciences, East Tower, Cardiovascular Research Inst., University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; :verkman{at}itsa.ucsf.edu.

(^1)
The abbreviations used are: MIP, major intrinsic protein; IMP, intramembrane particle; OAP, orthogonal arrays of particles; MIWC, mercurial insensitive water channel; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Javier Farinas for protein sequence calculations and Tonghui Ma for the plasmid construction.


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