Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70: 69: 29 wild type: heterozygote: knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew; 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wildtype mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.

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.

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

The water and solute transporting properties of the epidermis have been proposed to be important determinants of skin moisture content and barrier properties. The water/small solute-transporting protein aquaporin-3 (AQP3) was found by immunofluorescence and immunogold electron microscopy to be expressed at the plasma membrane of epidermal keratinocytes in mouseskin. We studied the role of AQP3 in stratum corneum (SC) hydration by comparative measurements in wildtype and AQP3 null mice generated in a hairless SKH1 genetic background. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries but were polyuric because of defective urinary concentrating ability. AQP3 deletion resulted in a >4-fold reduced osmotic water permeability and >2-fold reduced glycerol permeability in epidermis. Epidermal, dermal, and SC thickness and morphology were not grossly affected by AQP3 deletion. Surface conductance measurements showed remarkably reduced SC water content in AQP3 null mice in the hairless genetic background (165