©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of an Aquaporin cDNA from Salivary, Lacrimal, and Respiratory Tissues (*)

(Received for publication, September 8, 1994; and in revised form, October 24, 1994)

Surabhi Raina (§) Gregory M. Preston William B. Guggino Peter Agre (¶)

From the Departments of Biological Chemistry, Medicine, and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Aquaporin family of water channels plays a fundamental role in transmembrane water movements in numerous plant and animal tissues. Since the molecular pathway by which water is secreted by salivary glands is unknown, a cDNA was isolated from rat submandibular gland by homology cloning. Similar to other Aquaporins, the salivary cDNA encodes a 265-residue polypeptide with six putative transmembrane domains separated by five connecting loops (A-E); the NH(2)- and COOH-terminal halves of the polypeptide are sequence-related, and each contains the motif Asn-Pro-Ala. A mercurial-inhibition site is present in extracellular loop E, and cytoplasmic loop D contains a cAMP-protein kinase phosphorylation consensus. In vitro translation yielded a 27-kDa polypeptide, and expression of the cRNA in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability (P) which was reversibly inhibited by 1 mM HgCl(2). Northern analysis demonstrated a 1.6-kilobase mRNA in submandibular, parotid, and sublingual salivary glands, lacrimal gland, eye, trachea, and lung. In situ hybridization revealed a strong hybridization over the corneal epithelium in eye and over the secretory lobules in salivary glands. These studies have identified a new mammalian member of the Aquaporin water channel family (gene symbol AQP5) which is implicated in the generation of saliva, tears, and pulmonary secretions.


INTRODUCTION

Members of the Aquaporin family of membrane water channels exist throughout nature (reviewed by Chrispeels and Agre(1994)). Channel-forming integral protein (CHIP) was the first recognized Aquaporin (reviewed by Agre et al., 1993a). CHIP has been purified from red cells and renal proximal tubules (Denker et al., 1988; Smith and Agre, 1991); the complementary DNA (cDNA) was isolated from an erythroid library (Preston and Agre, 1991) and is encoded by the single copy gene Aquaporin-1, AQP1 (Moon et al., 1993). CHIP was first functionally defined as a molecular water channel by expression in Xenopus oocytes (Preston et al., 1992), and the function was confirmed with proteoliposomes containing the pure protein (Zeidel et al., 1992).

Immunohistochemical studies using affinity-purified antibodies to either the NH(2) or COOH terminus of CHIP showed the protein to be abundant in renal proximal tubules and descending thin limbs of Henle's loop (Denker et al., 1988, Sabolic et al., 1992, Nielsen et al., 1993a) where it is expressed at discrete stages during fetal development (Bondy et al., 1993; Smith et al., 1993; Agre et al., 1994). CHIP protein was also found in several other water-permeable tissues including choroid plexus, ciliary epithelium, lens epithelium, corneal endothelium, hepatobiliary epithelium, capillary endothelium (Nielsen et al., 1993b), and male reproductive tract (Brown et al., 1993). Functional studies have implicated CHIP in transmembrane water movements of red cells (Zeidel et al., 1992; Van Hoek and Verkman 1992; Preston et al., 1994a), renal tubules (Echevarria et al., 1993a; Zhang et al., 1993), corneal endothelium (Echevarria et al., 1993b), alveolar epithelium (Folkesson et al., 1994), and hepatobiliary epithelium (Roberts et al., 1994). Surprisingly, no clinical phenotype was observed in humans homozygous for Aquaporin-1 knockout mutations, suggesting that other water channels must exist (Preston et al., 1994a).

The sequences of CHIP (Preston and Agre, 1991) and the functionally undefined homolog MIP, major intrinsic protein of lens (Gorin et al., 1984), have led to homology cloning of other members of the Aquaporin family (reviewed by Knepper(1994)). AQP-CD is a water channel protein expressed in renal collecting ducts and is encoded by Aquaporin-2 (Fushimi et al., 1993; Sasaki et al., 1994). Antidiuretic hormone regulates the subcellular distribution of this protein (Nielsen et al., 1993c; DiGiovanni et al., 1994), and Aquaporin-2 is the site of mutations in some forms of nephrogenic diabetes insipidus (Deen et al., 1994). Aquaporin-3 encodes a water channel protein which is also permeable to glycerol and is located in the basolateral membranes of renal medullary collecting duct and intestine (Ishibashi et al., 1994). A mercury-insensitive water channel was identified (Hasegawa et al., 1994), and Aquaporin-4 is most abundantly expressed in brain where it has been implicated as the hypothalamic osmoreceptor which mediates vasopressin secretion (Jung et al., 1994a).

The molecular pathways through which salivary and lacrimal glands secrete water are not yet explained (reviewed by Nauntofte, 1992), and none of the known members of the Aquaporin family exist at these locations nor in large airway epithelium (Nielsen et al., 1993b; Li et al., 1994). Therefore, other Aquaporins may be involved in several important clinical settings. For example, patients with Sjögren's syndrome suffer from the lack of normal tear and saliva secretion due to autoimmune destruction of lacrimal and salivary glands, yet the target antigen in the glandular epithelia is not known (Fox and Saito, 1994). Although patients with head and neck cancer may be cured of their malignancies, they often suffer from inanition and aspiration pneumonias due to the inability to secrete saliva after local radiotherapy (Vokes et al., 1993). Dryness of eyes is a common and unexplained aging phenomenon which can lead to loss of vision in some individuals (Greiner and Kenyon, 1994). Evaporation of water from the large airways is thought to precipitate asthma (McFadden and Gilbert, 1994). Here we report the isolation and molecular characterization of the 5th mammalian member of the Aquaporin family which is expressed at locations implicating it in the generation of saliva, tears, and pulmonary secretions.


MATERIALS AND METHODS

cDNA Cloning

Rat submandibular gland mRNA (1 µg) was reverse-transcribed using random hexamer primers and reverse transcriptase (Perkin-Elmer Cetus). Nested, degenerate oligonucleotide primers were designed corresponding to the most highly conserved sequences surrounding the NPA motifs in the Aquaporins (Reizer et al., 1993; Preston, 1993): sense primers MDU-1 (5`-STBGGNCAYRTBAGYGGNGC-NCA-3`) and MDU-2 (5`GGGATCCGCHCAYNTNAAYCCHG-YN-GTNAC-3`), antisense primers MDD-1 (5`-GCDGRNSCVARDGANCGNGCNGG-3`) and MDD-2 (5`-CGGAATTCGDGCDGGRTTNATNSHNSMNCC-3`). The reverse-transcribed RNA was amplified by 30 cycles of polymerase chain reaction (1 min at 94 °C, 1 min at 52 °C, 1 min at 72 °C) using 100 pmol of MDU-1 and MDD-1; the products were reamplified using 100 pmol of MDU-2 and MDD-2. A 376-bp (^1)product was ligated into the EcoRV site of pBS-KS(+) for bacterial transformation. This DNA fragment was radiolabeled with [alpha-P]dCTP (3,000 Ci/mmol, Amersham) and used to probe nylon membranes (Colony/Plaque Screen, DuPont NEN) containing 2 times 10^5 plaques from an adult Sprague-Dawley rat submandibular gland cDNA library in ZAP II (Girard et al., 1993). Membranes were hybridized for 18 h with 10^6 cpm/ml of probe, and washed at 65 °C in 0.2 times SSC, 0.1% SDS. A 1.5-kb insert from a purified plaque was subcloned for double-stranded dideoxynucleotide sequencing with Sequenase 2.0 (U.S. Biochemical) and [alpha-S]dATP (1,000 Ci/mmol).

Expression in Vitro

Protein synthesis from the salivary cDNA was demonstrated with a cell-free translation system (Promega). In vitro-transcribed cRNA (200 ng) was added to a rabbit reticulocyte lysate mixture containing [S]methionine and canine pancreatic microsomes. The reaction was incubated for 1 h at 30 °C, and the microsomes were pelleted at 100,000 times g for analysis by SDS-PAGE autoradiography.

Functional Expression in Oocytes

A 1-kb BamHI fragment containing the 5`-untranslated sequence and the entire open reading frame of the salivary cDNA was blunt-end-ligated into the BglII site of the Xenopus expression construct pXbetaG, which contains the HindIII-PstI insert of pSP64T in pBS II KS (Preston et al., 1992). Capped cRNA was synthesized in vitro after digestion with XbaI (Preston et al., 1993). Defolliculated stage V-VI Xenopus oocytes were injected with 50 nl of water or up to 5 ng of sample cRNAs and incubated for 3 days at 18 °C in 200 mosM modified Barth's buffer. The oocytes were transferred to 70 mosM modified Barth's buffer at 21 °C; oocyte swelling was monitored by videomicroscopy, and the coefficient of osmotic water permeability (P(f)) was determined (Preston et al., 1992, 1993). Uptake of [^14C]urea or [^14C]glycerol was measured by incubating oocytes in 200 mosM Barth's buffer for 10-20 min at 21 °C followed by washing and SDS solubilization. No increase in ion conductance was found when control oocytes were compared to RNA-injected oocytes by two electrode voltage clamps (Preston et al., 1992).

Northern Blots

RNA was isolated from various rat tissues using RNazol B solution (Cinna Scientific). Aliquots of total RNA or poly(A) RNA were resolved on a formaldehyde agarose gel, transferred to nylon membranes, and hybridized at high stringency with the full-length salivary cDNA probe labeled with [alpha-P]dCTP as described (Preston and Agre, 1991).

In Situ Hybridizations

Cryosections of 12 µm were cut from rat submandibular gland and eye, fixed with 4% paraformaldehyde in phosphate-buffered saline, and treated with 0.25% acetic anhydride, 0.1 M triethanolamine for 10 min. [S]UTP-labeled antisense and sense riboprobes were made with T(7) or T(3) RNA polymerase from the 1-kb BamHI DNA fragment (see above). Sections were hybridized overnight at 56 °C with 10^6 cpm of probe. After RNase treatment, sections were exposed to autoradiographic film for 1 day and to photographic emulsion for 4 days (Bhat et al., 1994).


RESULTS AND DISCUSSION

Isolation of a cDNA from Salivary Gland

The most conserved sequences of the Aquaporins were used to design degenerate oligonucleotide primers, and a cDNA library from rat submandibular gland was the template for polymerase chain amplification. The 376-bp product was used to isolate two positive plaques from the same library (see ``Materials and Methods''). The 1.5-kb insert contained a 109-bp 5`-untranslated sequence preceding an initiation site consensus (Kozak, 1987). A 795-bp open reading frame was followed by 3`-untranslated sequence containing a polyadenylation consensus (Fig. 1A).


Figure 1: Sequence analysis and predicted topology of salivary cDNA, AQP5. A, nucleotide sequence and deduced amino acid sequence of the clone isolated from a rat submandibular gland cDNA library. Presumed bilayer spanning domains are underlined, and adjacent putative N-glycosylation sites are identified (*). Conserved NPA motifs and mammalian cAMP-protein kinase motifs are enclosed. A polyadenylation consensus is double-underlined. B, hydropathy analysis of deduced amino acid sequence using a 7-residue window (Kyte and Doolittle, 1982). C, proposed membrane topology based upon sequence analysis and hourglass model for Aquaporins (Preston et al., 1994b; Jung et al., 1994b) comprised of six bilayer-spanning domains and five connecting loops (A-E). Domains with 40-65% of residues identical with AQP1 (CHIP) are represented as heavy lines (bilayer spanning domains and loops B, D, and E); domains and loops with leq25% identity are thin lines (NH(2)- and COOH termini; loops A and C). Locations of selected residues are identified: N-glycosylation site, NPA motifs in the first and second aqueous hemichannels, residues corresponding to mercury-sensitive sites in AQP1, cAMP-protein kinase motif.



Deduced Structure of the Salivary Polypeptide

Analysis of the GenBank data base revealed the salivary cDNA to be a novel member of the Aquaporin family. The open reading frame encodes a polypeptide of 265 amino acids. Hydropathy analysis confirmed six putative bilayer-spanning domains and five connecting loops of which loops B and E were also hydrophobic (Fig. 1B). Loops B and E each contained the recognized NPA motif (Asn-Pro-Ala) present in all Aquaporins; the 2nd NPA was flanked by a cysteine at residue 182 (Fig. 1C) corresponding to the known mercurial-inhibitory site of AQP1 (Preston et al., 1993). The deduced protein was 45% identical overall with AQP1, with the highest levels of homology in the six bilayer-spanning domains and loops B, D, and E (Fig. 1C). Two N-glycosylation consensus sites were identified at residues Asn-124 and Asn-125 in loop C, supporting the extracellular location of this domain as predicted by topology mapping (Preston et al., 1994b) and consistent with potential N-glycosylation sites in AQP2 (Fushimi et al., 1993), AQP3 (Ishibashi et al., 1994), and AQP4 (Hasegawa et al., 1994; Jung et al., 1994a). Although cytoplasmic loop D is predicted to be only 8 residues long, it contains the sequence Ser-Arg-Arg-Thr-Ser, matching the consensus for cAMP-protein kinase phosphorylation (Kennelly and Krebs, 1991), a motif present also in the COOH-terminal domain of AQP2 (Fushimi et al., 1993).

Expression and Transport Function

Cell-free translation of the cRNA encoding AQP1 (CHIP) in the presence of microsomes yielded a band at 28 kDa by SDS-polyacrylamide gel electrophoresis and slightly larger bands corresponding to glycosylated polypeptides; similar translation of the salivary cRNA yielded a major band at 27 kDa (Fig. 2A). This corresponds to the predicted mass of 28.4 kDa with slightly more rapid electrophoretic mobility probably resulting from a larger number of hydrophobic residues (Helenius and Simons, 1975).


Figure 2: Expression of AQP5 and water permeability determinations. A, cell-free expression in the presence of microsomes of AQP1 and AQP5 cRNAs. B, osmotic water permeability (P) of oocytes injected with 50 nl of water without RNA or oocytes injected with 5 ng of the indicated cRNAs. Shown are the mean values and standard deviations of 4-5 oocytes receiving no further treatment (stippled bars), oocytes incubated for 5 min in 1 mM HgCl(2) (black bars), or oocytes incubated for 5 min in 1 mM HgCl(2) followed by 30 min in 5 mM beta-mercaptoethanol (open bars).



Transmembrane water flow through the salivary protein was evaluated by expression in Xenopus oocytes. After 3 days of incubation at 18 °C, the oocytes were transferred from 200 mosM to 70 mosM modified Barth's solution; swelling was monitored by videomicroscopy, and the coefficient of osmotic water permeability (P(f)) was calculated. Similar to oocytes injected with AQP1 cRNA, injection of oocytes with the salivary cRNA increased the P(f) by approximately 20-fold (Fig. 2B). Therefore, the salivary protein qualifies as the 5th mammalian member of the Aquaporin family of water channels (Agre et al., 1993b), and the gene has been designated Aquaporin-5 (symbol AQP5) by the Genome Data Base.

The increase in P(f) mediated by expression of AQP1 is blocked by treatment with 1 mM HgCl(2) and restored by incubation in beta-mercaptoethanol (Fig. 2B; Preston et al., 1992). Similar treatment of oocytes expressing AQP5 with 1 mM HgCl(2) produced a comparable reduction in P(f) which was restored by incubation with beta-mercaptoethanol (Fig. 2B). Likewise, oocytes expressing AQP5 exhibited no increase in the membrane transport of [^14C]urea or [^14C]glycerol above that of water-injected oocytes, and electrophysiological studies failed to reveal increased membrane conductance (not shown).

Tissue Distribution of AQP5 mRNA

Total RNA or poly(A) RNA from rat salivary glands showed a single prominent band of approximately 1.6 kb, and analyses of RNA from lacrimal gland, trachea, eye, and distal lung were similar. In contrast, no signal was detected with the AQP5 probe when comparable amounts of RNA from kidney, brain, intestine, or other tissues were analyzed (Fig. 3).


Figure 3: Northern analysis of AQP5 mRNA in rat tissues. Total RNA (10 µg) or poly(A) RNA (1 µg) from submandibular gland, sublingual gland, and other indicated rat tissues hybridized with P-labeled probe corresponding to full-length AQP5 cDNA. Equivalent amounts of RNA were loaded as verified by the abundance of 18 S and 28 S RNAs and Northern analysis using a mouse -actin probe (not shown).



In situ hybridizations of eye with an antisense AQP5 riboprobe revealed a discrete pattern of expression over the corneal epithelium, whereas the sense riboprobe gave a negligible signal (Fig. 4A). This pattern of localization contrasted with that of other Aquaporin transcripts in eye. Corneal endothelium, lens epithelium, and nonpigmented epithelium of ciliary body and iris are known to express AQP1 (Nielsen et al., 1993b); however, none were found to express AQP5 (Fig. 4A and other data not shown). Lens fiber cells are known to express large amounts of the homolog major intrinsic protein, MIP (Gorin et al., 1984), but no AQP5 hybridization was detected (not shown). Neither the retinal neuronal nuclear layer, which expresses AQP4 (Hasegawa et al., 1994), nor retinal pigmented epithelium gave specific AQP5 signals (not shown).


Figure 4: In situ localization of AQP5 mRNA in rat eye and submandibular salivary gland. A-A`, bright field and dark field views of a section of eye examined by in situ hybridization with antisense probe revealing intense signal over corneal epithelium (epi) but not over corneal endothelium (end), ciliary body (cil), iris (ir), or other structures. B-B`, bright field and dark field views of a section of submandibular gland examined by in situ hybridization with antisense probe revealing intense signal over secretory lobules (lob) but not over excretory ducts (duc) or stromal septae (sep). Parallel incubations with sense probes failed to demonstrate hybridization in cornea or submandibular gland even after prolonged autoradiographic exposure (insets). Magnifications times 45.



In situ hybridizations of salivary glands also revealed discrete hybridization patterns. Submandibular glands contain serous and mucus-secreting epithelia within the glandular lobules. Strong hybridization was identified over the secretory lobules, whereas only background levels of hybridization were detected over the stromal septae and excretory ducts (Fig. 4B). Specificity of these hybridizations was confirmed by control studies with sense riboprobes which failed to hybridize (Fig. 4B, inset). Distribution of AQP5 mRNA was similar in parotid and sublingual salivary glands and lacrimal glands (not shown).

Physiological Roles and Potential Regulatory Mechanisms

As first established in kidney (reviewed by Nielsen and Agre(1995)), other organs also express multiple Aquaporins, since eye expresses at least three (AQP1, AQP4, and AQP5) in addition to the functionally undefined homolog, MIP. The localization of each is highly specific, with expression of different homologs in adjacent tissues (e.g. corneal epithelium and endothelium, lens epithelium, and fiber cells). No tissue has been found with overlapping expression of two or more different Aquaporins at the same cellular location, implying that each plays a particular role in vision. Our studies predict that AQP5 is involved in lacrimation and corneal desiccation, consistent with the presence of water channels demonstrated in these locations (Candia and Zamudio, 1995). Previous studies documented expression of AQP1 (CHIP) in the endothelium of capillaries in the soft tissues surrounding salivary glands, but absence of the protein from the gland (Nielsen et al., 1993b, Li et al., 1994). This suggests that water drawn from surrounding capillaries is secreted by the glandular epithelium and is not reabsorbed in excretory ducts. Although Northern blots of trachea and distal lung both contained strong AQP5 signals (Fig. 3), the specific distribution was not resolved by in situ hybridization and will require immunohistochemical analysis.

The driving force for water movement is presumably provided by the osmotic gradients which result from evaporation of water from the cornea or transport of ions by glandular epithelia (reviewed by Nauntofte(1992)). Nevertheless, a role for adrenergic regulation of AQP5 must be considered, since salivation and lacrimation are known to be controlled by the autonomic nervous system (reviewed by Baum (1987)). Sequence comparisons of the mammalian Aquaporins revealed that AQP5 is least closely related to AQP1 and AQP3 whose products are thought to be constitutively active water channels; in contrast, AQP5 is most closely related to AQP2 (Fig. 5). The cellular distribution of AQP2 is known to be regulated by antidiuretic hormone which increases cellular cAMP levels (Nielsen et al., 1993c), and the COOH terminus of AQP2 contains a cAMP-protein kinase consensus identical with that within loop D of AQP5. It is not clear whether the cAMP-protein kinase consensus on AQP2 is required for this response or whether direct phosphorylation of the channel can modulate its activity, but we speculate that AQP5 is regulated by an adrenergic hormone-cAMP cascade analogous to that of antidiuretic hormone and AQP2. The deduced amino sequence for AQP5 should permit development of specific reagents needed to test this hypothesis.


Figure 5: Sequence comparison of members of the mammalian Aquaporin protein family. The amino acid sequences of human AQP1 (Preston and Agre, 1991), bovine MIP (Gorin et al., 1984), and rat AQP2 (Fushimi et al., 1993), AQP3 (Ishibashi et al., 1994), AQP4 (Jung et al., 1994a), and AQP5 (Fig. 1) were aligned by the PILEUP program (version 7.1, Genetic Computer Group, Madison WI) of progressive alignments (Feng and Doolittle, 1990) using a gap weight of 3.0 and gap length of 0.1 running on a VAX computer system. The percentage amino acid identity between AQP5 and each of the other Aquaporins is represented.




FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL33991 and HL48268 (to P. A.) and DK32753 (to W. B. G.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16245[GenBank].

§
Present address: Dept. of Embryology, Carnegie Institution of Washington, 115 W. University Parkway, Baltimore, MD 21210.

To whom correspondence and reprint requests should be addressed: Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Fax: 410-955-3149.

(^1)
The abbreviations used are: bp, base pair(s); kb, kilobase(s); P, coefficient of osmotic water permeability.


ACKNOWLEDGEMENTS

We thank Lilly Mirels, Julie Reeves, Ratan Bhat, Tiziana P. Carroll, and Landon King for assistance and helpful discussions.


REFERENCES

  1. Agre, P., Preston, G. M., Smith, B. L., Jung, J. S., Raina, S., Moon, C., Guggino, W. B., and Nielsen, S. (1993a) Am. J. Physiol. 265, F463-F476
  2. Agre, P., Sasaki, S., and Chrispeels, M. J. (1993b) Am. J. Physiol. 265, F461
  3. Agre, P., Smith, B. L., Baumgarten, R., Preston, G. M., Pressman, E., Wilson, P., Illum, N., Anstee, D. J., Lande, M. B., and Zeidel, M. L. (1994) J. Clin. Invest. 94, 1050-1058 [Medline] [Order article via Infotrieve]
  4. Baum, B. J. (1987) J. Dent. Res. 66, 628-632 [Medline] [Order article via Infotrieve]
  5. Bhat, R. V., Baraban, J. M., Johnson, R. C., Eipper, B. A., and Mains, R. E. (1994) J. Neurosci. 14, 3059-3071 [Abstract]
  6. Bondy, C., Chin, E., Smith, B. L., Preston, G. M., and Agre, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4500-4504 [Abstract]
  7. Brown, D., Verbavatz, J. M., Valenti, G., Lui, B., and Sabolic, I. (1993) Eur. J. Cell Biol. 61, 264-273 [Medline] [Order article via Infotrieve]
  8. Candia, O. A., and Zamudio, A. C. (1995) J. Membr. Biol. , in press
  9. Chrispeels, M. J., and Agre, P. (1994) Trends Biochem. Sci. 19, 421-425 [CrossRef][Medline] [Order article via Infotrieve]
  10. Deen, P. M. T., Verdijk, M. A. J., Knoers, N. V. A. M., Wieringa, B., Monnens, L. A. H., van Os, C. H., and van Oost, B. A. (1994) Science 264, 92-95 [Medline] [Order article via Infotrieve]
  11. Denker, B. M., Smith, B. L., Kuhajda F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634-15642 [Abstract/Free Full Text]
  12. DiGiovanni, S. R., Nielsen, S., Christensen, E. I., and Knepper, M. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8984-8988 [Abstract]
  13. Echevarria, M., Frindt, G., Preston, G. M., Milovanovic, S., Agre, P., Fischbarg, J., and Windhager, E. (1993a) J. Gen. Phys. 101, 827-841 [Abstract]
  14. Echevarria, M., Kuang, K., Iserovich, P., Li, J., Preston, G. M., Agre, P., and Fischbarg, J. (1993b) Am. J. Physiol. 265, C1349-C1355
  15. Feng, D. F., and Doolittle, R. F. (1990) Methods Enzymol. 183, 375-387 [Medline] [Order article via Infotrieve]
  16. Folkesson, H. G., Matthay, M. A., Hasegawa, G., Kheradmand, F., and Verkman, A. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4970-4974 [Abstract]
  17. Fox, R. I., and Saito, I. (1994) Arthritis Rheum. 37, 771-772 [Medline] [Order article via Infotrieve]
  18. Fushimi, K., Uchida, S., Hara, Y., Hirata, Y., Marumo, F., and Sasaki, S. (1993) Nature 361, 549-552 [CrossRef][Medline] [Order article via Infotrieve]
  19. Girard, L. R., Castle, A. M., Hand, A. R., Castle, J. D., and Mirels, L. (1993) J. Biol. Chem. 268, 26592-26601 [Abstract/Free Full Text]
  20. Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.-P., and Horwitz, J. (1984) Cell 39, 49-59 [Medline] [Order article via Infotrieve]
  21. Greiner, J. V., and Kenyon, K. R. (1994) in Principles and Practice of Ophthalmology-Basic Sciences (Albert, D. M., and Jokobiec, F. A., eds) Chap. 52, W. B. Saunders Co., Philadelphia
  22. Hasegawa, H., Ma, T., Skach, W., Matthay, M. A., and Verkman, A. S. (1994) J. Biol. Chem. 269, 5497-5500 [Abstract/Free Full Text]
  23. Helenius, A., and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79 [Medline] [Order article via Infotrieve]
  24. Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwahara, M., Saito, H., Furukawa, T., Nakajima, K., Yamaguchi, Y., Gojobori, T., and Marumo, F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6269-6273 [Abstract]
  25. Jung, J. S., Bhat, R. V., Preston, G. M., Guggino, W. B., Baraban, J. M., and Agre, P. (1994a) Proc. Natl. Acad. Sci. U. S. A. 91, 13052-13056 [Abstract/Free Full Text]
  26. Jung, J. S., Preston, G. M., Smith, B. L., Guggino, W. B., and Agre, P. (1994b) J. Biol. Chem. 269, 14648-14654 [Abstract/Free Full Text]
  27. Kennelly, P. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 15555-15558 [Free Full Text]
  28. Knepper, M. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6255-6258 [Free Full Text]
  29. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8132 [Abstract]
  30. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 105, 105-132
  31. Li J., Nielsen, S., Dai, Y., Lasowski, K. W., Christensen, E. I., Tabak, L. A., and Baum, B. J. (1994) Pflügers Arch 428, 455-460
  32. McFadden, E. R., and Gilbert, I. A. (1994) N. Engl. J. Med. 330, 1362-1367 [Free Full Text]
  33. Moon, C., Preston, G. M., Griffin, C. A., Jabs, E. W., and Agre, P. (1993) J. Biol. Chem. 268, 15772-15778 [Abstract/Free Full Text]
  34. Nauntofte, B. (1992) Am J. Physiol. 263, G823-G837
  35. Nielsen, S., and Agre, P. (1995) Kidney Int. , in press
  36. Nielsen, S., Smith, B. L., Christensen, E. I., Knepper, M. A., and Agre, P. (1993a) J. Cell Biol. 120, 371-383 [Abstract]
  37. Nielsen, S., Smith, B. L., Christensen, E. I., and Agre, P. (1993b) Proc. Natl. Acad. Sci. U. S. A. 90, 7275-7279 [Abstract]
  38. Nielsen, S., DiGiovanni, S. R., Christensen, E. I., Knepper, M. A., and Harris, H. W. (1993c) Proc. Natl. Acad. Sci. U. S. A. 90, 11663-11667 [Abstract]
  39. Preston, G. M. (1993) in Methods in Molecular Biology (White, B. A., ed) Vol. 15, pp. 317-337, Humana Press, Totowa, NJ _
  40. Preston, G. M., and Agre, P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11110-11114 [Abstract]
  41. Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387 [Medline] [Order article via Infotrieve]
  42. Preston, G. M., Jung, J. S., Guggino, W. B., and Agre, P. (1993) J. Biol. Chem. 268, 17-20 [Abstract/Free Full Text]
  43. Preston, G. M., Smith, B. L., Zeidel, M. L., Moulds, J., and Agre, P. (1994a) Science 265, 1585-1587 [Medline] [Order article via Infotrieve]
  44. Preston, G. M., Jung, J. S., Guggino, W. B., and Agre, P. (1994b) J. Biol. Chem. 269, 1668-1673 [Abstract/Free Full Text]
  45. Reizer, J., Reizer, A., and Saier, M. H., Jr. (1993) Crit. Rev. Biochem. Mol. Biol. 28, 235-257 [Abstract]
  46. Roberts, S. K., Yano, M., Ueno, Y., Pham, L., Alpini, G., Agre, P., and LaRusso, N. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 13009-13013 [Abstract/Free Full Text]
  47. Sabolic, I., Valenti, G., Verbavatz, J. M., van Hoek, A. N., Verkman, A. S., Ausiello, D. A., and Brown, D. (1992) Am. J. Physiol. 263, C1225-C1233
  48. Sasaki, S., Fushimi, K., Saito, H., Saito, F., Uchida, S., Ishibashi, K., Kuwahara, M., Ikeuchi, T., Inui, K., Nakajima, K., Watanabe, T. X., and Marumo, F. (1994) J. Clin. Invest. 93, 1250-1256 [Medline] [Order article via Infotrieve]
  49. Smith, B. L., and Agre, P. (1991) J. Biol. Chem. 266, 6407-6415 [Abstract/Free Full Text]
  50. Smith, B. L., Baumgarten, R., Nielsen, S., Raben, D., Zeidel, M. L., and Agre, P. (1993) J. Clin. Invest. 92, 2035-2041 [Medline] [Order article via Infotrieve]
  51. Van Hoek, A. N., and Verkman, A. S. (1992) J. Biol. Chem. 267, 18267-18269 [Abstract/Free Full Text]
  52. Vokes, E. E., Weichselbaum, R. R., Lipman, S. M., and Hong, W. K. (1993) N. Engl. J. Med. 328, 184-194 [Free Full Text]
  53. Zeidel, M. L., Ambudkar, S. V., Smith, B. L., and Agre, P. (1992) Biochemistry 31, 7436-7440 [Medline] [Order article via Infotrieve]
  54. Zhang, R., Skach, W., Hasegawa, H., van Hoek, A. N., and Verkman, A. S. (1993) J. Cell Biol. 120, 359-369 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.