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

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