(Received for publication, September 8, 1994; and in revised form, October 24, 1994)
From the
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- 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
. 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.
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 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.
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 25%
identity are thin lines (NH
- 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.
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
(black bars), or oocytes incubated for 5
min in 1 mM HgCl
followed by 30 min in 5 mM
-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)
was calculated. Similar to oocytes injected with AQP1 cRNA,
injection of oocytes with the salivary cRNA increased the P
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 mediated by expression of AQP1 is blocked by treatment with 1 mM HgCl
and restored by incubation in
-mercaptoethanol (Fig. 2B; Preston et al., 1992). Similar
treatment of oocytes expressing AQP5 with 1 mM HgCl
produced a comparable reduction in P
which was restored by incubation with
-mercaptoethanol (Fig. 2B). Likewise, oocytes
expressing AQP5 exhibited no increase in the membrane
transport of [
C]urea or
[
C]glycerol above that of water-injected
oocytes, and electrophysiological studies failed to reveal increased
membrane conductance (not shown).
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
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).
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16245[GenBank].