Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid wateryglycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-D-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to 30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3yAQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.

Aquaporin-4 (AQP4) is a mercurial-insensitive, water-selective channel that is expressed in astroglia and basolateral plasma membranes of epithelia in the kidney collecting duct, airways, stomach, and colon. A targeting vector for homologous recombination was constructed using a 7-kb SacI AQP4 genomic fragment in which part of the exon 1 coding sequence was deleted. Analysis of 164 live births from AQP4[+/-] matings showed 41 [-/-], 83 [+/-], and 40 [-/-] genotypes. The [-/-] mice expressed small amounts of a truncated AQP4 transcript and lacked detectable AQP4 protein by immunoblot analysis and immunocytochemistry. Water permeability in an AQP4-enriched brain vesicle fraction in [+/+] mice was high and mercurial insensitive, and was decreased by 14-fold in [-/-] mice. AQP4 deletion did not affect growth or tissue morphology at the light microscopic level. Northern blot analysis showed that tissue-specific expression of AQPs 1, 2, 3, and 5 was not affected by AQP4 deletion. Maximum urine osmolality after a 36-h water deprivation was (in mosM, n=15) [+/+] 3,34

In searching for a basolateral membrane water trans-porter in rat kidney with homology to channel formingintegral protein (CHIP28), water channel-collecting duct (WCH-CD), and mercurial-insensitive water chan-nel (MIWC), we cloned a new member of the major in-trinsic protein family (GLIP, -cero1 Intrinsic B o -tein). GLIP cDNA had an 865-base pair open reading frame encoding a 30.6-kDa protein with 19-23% amino acid identity to the water channels and 36% identity to the bacterial glycerol facilitator GlpF. Northern blot analysis showed a 6.6-kilobase mFWA encoding GLIP in kidney, brain, and lung; RT-PCWSouthern blot analysis indicated expression of GLIP in kidney, brain, lung, eye, colon, stomach, and skeletal muscle, but not in heart, liver, and spleen. In situ hybridization in rat kidney showed GLIP mRNA expression in medullary collecting duct. Immunofluorescence with a peptide-derived poly-clonal antibody showed GLIP protein expression in ba-solateral membrane of kidney collecting duct principal cells and brain meningeal cells. Functional measure-ments in Xenopus oocytes expressing GLIP cRNA showed a>2O-fold increase in [SHlglycerol uptake com-pared with water-iqjected oocytes; glycerol uptake was inhibited 88% by diisothiocyanodisdfonic stilbene (0.2mm) and 36% by phloretin (0.25mm). GLIP did not func-tion as a transporter for water, urea, inositol, glucose, lactate, and monovalent ions. Glycerol uptake in oocytes expressing CHIP28 and MIWC was not different from that in water-injected controls. GLIP represents the first mammalian water channel homolog that selectively transports a solute other than water. The physiological substrate(s) and role(s) of GLIP remain to be elucidated.

T1 a is a protein of unknown function that is expressed at the plasma membrane in epithelia involved in fluid transport, including type I alveolar epithelial cells, choroid plexus, and ciliary epithelium. The purpose of this study was to test the hypothesis that T1 a functions as a water channel or a regulator of aquaporin-type water channels that are coexpressed with T1 a. Two complementary DNAs (cDNAs)(hT1a-1 and hT1 a-2) encoding human isoforms of T1 a were cloned by homology to the rat T1 a coding sequence. The cDNAs encoded 164 (hT1 a-1) and 162 (hT1 a -2) amino acid proteins with high homology to rat T1 a in a putative membrane-spanning domain. hT1 a-1 transcripts of 2.6 and 1.4 kb were detected in human lung, heart, and skeletal muscle, and a single hT1 a-2 transcript of 1.2 kb was detected in human lung. Rat and mouse T1 a were isolated by reverse transcription-polymerase chain reaction and confirmed by DNA sequence analysis. Expression of mouse, rat, and human T1 a isoforms in Xenopus oocytes did not increase osmotic water permeability (Pf) above that in water-injected oocytes, nor was there an effect of protein kinase A or C activation; Pf was increased. 10-fold in positive control oocytes expressing aquaporin (AQP)1 or AQP5. Coexpression of AQP1 or AQP5 with excess T1a gave P f not different from that in oocytes expressing AQP1 or AQP5 alone. Oocyte plasma membrane localization of epitope-tagged T1 a protein was confirmed and quantified by immunoprecipitation of microdissected plasma membranes. Quantitative densitometry indicated that the single-channel water permeability of T1 a is under 23102 16cm 3/s, suggesting that T1 a is not involved in the high transalveolar water permeability in intact lung. The cloning of hT1 a isoforms may permit the development of an assay of type I cell antigen in airspace fluid as a marker of human lung injury. Ma, T., B. Yang, M. A. Matthay, and A. S. Verkman. 1998. Evidence gainst a role of mouse, rat, and two cloned human T1 a isoforms as a water channel or a regulator of aquaporin-type water channels. Am. J. Respir. Cell Mol. Biol. 19: 143-149.

and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (Kd>10M). When added for 10min, none of the compounds activated Phe508-CFTR in transfected cells grown at 37℃ (with Phe508-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48h of growth at 27℃, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl conductance with Kd<1M. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.