Water channel aquaporin-1 (AQP1) is strongly expressed in kidney in proximal tubule and descending limb of Henle epithelia and in vasa recta endothelia. The grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) and in AQP4 knockout mice has suggested that aquaporins (other than the vasopressin-regulated water channel AQP2) may not be important in mammalian physiology. We have generated transgenic mice lacking detectable AQP1 by targeted gene disruption. In kidney proximal tubule membrane vesicles from knockout mice, osmotic water permeability was reduced 8-fold compared with vesicles from wild-type mice. Although the knockout mice were grossly normal in terms of survival, physical appearance, and organ morphology, they became severely dehydrated and lethargic after water deprivation for 36h. Body weight decreased by 35 6 2%, serum osmolality increased to>500mOsm, and urinary osmolality (657 659 mOsm) did not change from that before water deprivation. In contrast, wild-type and heterozygous mice remained active after water deprivation, body weight decreased by 20-22%, serum osmolality remained normal (310-330mOsm), and urine osmolality rose to>2500mOsm. Urine [Na1] in water-deprived knockout mice was<10mM, and urine osmolality was not increased by the V2 agonist DDAVP. The results suggest that AQP1 knockout mice are unable to create a hypertonic medullary interstitium by countercurrent multiplication. AQP1 is thus required for the formation of a concentrated urine by the kidney.

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.

CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proxi-mal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced -8 X 10’copies of CHIP28k protein/cell. Plasma membrane os-motic water permeability (P f), measured by stopped-flow light scattering, was 0.004cm/s in control (vec-tor-transfected) cells (10C) and 0.014cm/s in the CHIP28k-transfected cells. P f in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgC12. CHIP28k expression did not affect the transport of protons and the small polar non-lectrolytes urea and formamide. CHIP28k im-munoreactivity and function was then determined in subcellular fractions. P f in 6-carboxyfluorescein-la-beled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002cm/s (control cells) and 0.011cm/s (CHIP28k-transfected cells); Pt in transfected cells was inhibited by HgC12. Immuno-blotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (-5000 monomers/pm2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies es-tablish a stably transfected somatic cell line that strongly expresses functional CHIP28k water chan-nels. As in the original proximal tubule cells, the ex-pressed CHIP28k protein is a selective water channel that is functional in endocytic vesicles and the cell plasma membrane.

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