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.

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia, AQP4 in airway epithelia, and AQP5 at the apical plasma membrane in type I cells of alveolar epithelia. We previously studied the role of AQP1 and AQP4 in lung fluid transport using knockout mice. Here, we examined the role of AQP5 using AQP5 knockout mice, which were recently shown to manifest defective saliva secretion. AQP5 deletion did not affect lung morphology at the light microscopic level, nor did it affect the distribution or expression of aquaporins 1, 3, or 4. Airspace-capillary osmotic water permeability (Pf) was measured in isolated perfused lungs by pleural surface fluorescence and gravimetric methods. Pf was reduced 10-fold by AQP5 deletion and was further reduced by 2- to 3-fold in AQP1/AQP5 double-knockout mice. Hydrostatic lung edema in response to acute increases in pulmonary artery pressure was not affected by AQP5 deletion. Active alveolar fluid absorption was measured in an in situ lung model from the increase in concentration of a volume marker in an isosmolar alveolar instillate. Interestingly, fluid absorption did not differ in litter-matched AQP5 knockout mice, nor was there an effect of AQP5 deletion when fluid absorption was maximally stimulated by pretreatment of mice with keratinocyte growth factor. These results indicate that AQP5 is responsible for the majority of water transport across the apical membrane of type I alveolar epithelial cells. The unimpaired alveolar fluid clearance in AQP5-null mice indicates that high alveolar water permeability is not required for active, near-isosmolar fluid transport.

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl-transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTRinh-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with KI approximately 300nM. CFTRinh-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTRinh-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl-channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K+ channels, or a series of other transporters. A single intraperitoneal injection of CFTRinh-172 (250

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.

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.