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Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a 168-kD membrane-spanning glycoprotein containing regulatory and nucleotide binding domains [1]. CFTR functions [...], The cystic fibrosis gene product (CFTR) is a complex protein that function as an adenosine 3,5-monophosphate (cAMP)-stimulated ion channel and possibly as a regulator ofintracellular processes. In order to determine whether the CFTR molecule contains a functional aqueous pathway, anion, water, and urea transport were measured in Xenopus oocytes expressing CFTR. Cyclic AMP agonists induced a Cl- conductance of 94 microsiemens and an increase in water permeability of 4 x 10(-4) centimeter per second that was inhibited by a Cl- channel blocker and was dependent on anion composition. CFTR has a calculated single channel water conductance of 9 x 10(-13) cubic centimeter per second, suggestting a pore-like aqueous pathway. Oocytes expressing CFTR also showed cAMP-stimulated transport of urea but not the larger solute sucrose. Thus CFTR contains a cAMP-stimulated aqueous pore that can transport anions, water, and small solutes. The results also provide functional evidence for water movement through an ion channel.

The article discusses the synthesis of a cell-based fluorescence screen, which is based on an extracellular triazacryptand-based K sensor. The screen is shown to exhibit green fluorescence and is used for the assay of several [K.sup.+] channels and transporters.

The simulation and detection algorithms are developed for various modes of biologically relevant complex diffusive phenomena, including confined and convective diffusion, and diffusion in the presence of barriers, rafts and interparticle interactions. Few examples are illustrated to show the use of conventional vs. the new computational methods in detecting these membrane phenomena.

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 [+ or -] 5 [micro]m and [[Na.sup.+]] was 115 [+ or -] 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by ~40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. KEY WORDS: water permeability * trachea * airway surface liquid * ventilator * fluorescence microscopy

The airway surface liquid (ASL) is the thin layer of fluid coating the luminal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL osmolality include epithelial cell solute and water transport properties, evaporative water loss, and the composition of secreted fluids. We developed a noninvasive approach to measure ASL osmolality using osmotically sensitive 400-nm-diam liposomes composed of phosphatidylcholine/cholesterol/polyethylene glycol-phosphatidylcholine (1:0.3:0.08 molar ratio). Calcein was encapsulated in the liposomes at self-quenching concentrations (30 mM) as a volume-sensitive marker, together with sulforhodamine 101 (2 mM) as a volume-insensitive reference. Liposome calcein/sulforhodamine 101 fluorescence ratios responded rapidly ([is less than] 0.2 s) and stably to changes in solution osmolality. ASL osmolality was determined from calcein/sulforhodamine 101 fluorescence ratios after addition of microliter quantities of liposome suspensions to the ASL. In bovine airway epithelial cells cultured on porous supports at an air-liquid interface, ASL thickness (by confocal microscopy) was 22 [micro]m and osmolality was 325 [+ or -] 12 mOsm. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 55 [micro]m and osmolality was 330 [+ or -] 36 mOsm. ASL osmolality was not affected by pharmacological inhibition of CFTR in airway cell cultures or by genetic deletion of CFTR in knockout mice. ASL osmolality could be increased substantially to [is greater than] 400 mOsm by exposure of the epithelium to dry air; the data were modeled mathematically using measured rates of osmosis and evaporative water loss. These results establish a ratio imaging method to map osmolality in biological compartments. ASL fluid is approximately isosmolar under normal physiological conditions, but can become hyperosmolar when exposed to dry air, which may induce cough and airway reactivity in some patients. KEY WORDS: water permeability * cystic fibrosis * fluorescence self-quenching * trachea * CFTR

Wang, Kasper S., Tonghui Ma, Ferda Filiz, A. S. Verkman, and J. Augusto Bastidas. Colon water transport in transgenic mice lacking aquaporin-4 water channels. Am J Physiol Gastrointest Liver Physiol 279: G463-G470, 2000.--Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (-/-) mice. The transepithelial osmotic water permeability coefficient ([P.sub.f]) of in vivo perfused colon of +/+ mice, measured using the volume marker [sup.14]C-labeled polyethylene glycol, was 0.016 [+ or -] 0.002 cm/s. [P.sub.f] of proximal colon was greater than that of distal colon (0.020 [+ or -] 0.004 vs. 0.009 [+ or -] 0.003 cm/s, P [is less than] 0.01). [P.sub.f] was significantly lower in -/- mice when measured in full-length colon (0.009 [+ or -] 0.002 cm/s, P [is less than] 0.05) and proximal colon (0.013 [+ or -] 0.002 cm/s, P [is less than] 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. -/- mice (0.80 [+ or -] 0.01 vs. 0.81 [+ or -] 0.01), but there was a slightly higher water content in defecated stool from -/- mice (0.68 [+ or -] 0.01 vs. 0.65 [+ or -] 0.01, P [is less than] 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 [+ or -] 9 vs. 51 [+ or -] 8 [micro]l [multiplied by] [min.sup.-1] [multiplied by] [g.sup.-1]). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration. mercurial-insensitive water channel; water permeability; fecal dehydration

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux ([J.sub.v]) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. [J.sub.v] in wild-type mice varied linearly with osmotic gradient size (4.4 x [10.sup.-5] [cm.sup.3] [s.sup.-1] [mOsm.sup.-1]) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H20 outflow pressure, the filtration coefficient was 4.7 [cm.sup.3] [s.sup.-1] [mOsm.sup.-1] and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. [J.sub.v] were ([cm.sup.3] [s.sup.-1] [mOsm.sup.-1] x [10.sup.-5], SEM, n = 7-12 mice): 3.8 [+ or -] 0.4 (wild type), 0.35 [+ or -] 0.02 (AQP1 null), 3.7 [+ or -] 0.4 (AQP4 null), and 0.25 [+ or -] 0.01 (AQP1/AQP4 null). The significant reduction in [P.sub.f] in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 [+ or -] 0.2-fold (SEM, five mice) reduced [P.sub.f] in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish a simple gravimetric method to quantify osmosis and filtration in intact mouse lung and provide direct evidence for a contribution of the distal airways to airspace-to-capillary water transport. KEY WORDS: water permeability * airways * transgenic mouse * membrane transport * aquaporin-1

Verkman, A. S., and Alok K. Mitra. Structure and function of aquaporin water channels. Am. J. Physiol. Renal Physiol. 278: F13-F28, 2000.--The aquaporins (AQPs) are a family of small membrane-spanning proteins (monomer size ~30 kDa) that are expressed at plasma membranes in many cells types involved in fluid transport. This review is focused on the molecular structure and function of mammalian aquaporins. Basic features of aquaporin structure have been defined using mutagenesis, epitope tagging, and spectroscopic and freeze-fracture electron microscopy methods. Aquaporins appear to assemble in membranes as homotetramers in which each monomer, consisting of six membrane-spanning [Alpha]-helical domains with cytoplasmically oriented amino and carboxy termini, contains a distinct water pore. Medium-resolution structural analysis by electron cryocrystallography indicated that the six tilted helical segments form a barrel surrounding a central pore-like region that contains additional protein density. Several of the mammalian aquaporins (e.g., AQP1, AQP2, AQP4, and AQP5) appear to be highly selective for the passage of water, whereas others (recently termed aquaglyceroporins) also transport glycerol (e.g., AQP3 and AQP8) and even larger solutes (AQP9). Evidence for possible movement of ions and carbon dioxide through the aquaporins is reviewed here, as well as evidence for direct regulation of aquaporin function by posttranslational modification such as phosphorylation. Important unresolved issues include definition of the molecular pathway through which water and solutes move, the nature of monomer-monomer interactions, and the physiological significance of aquaporin-mediated solute movement. Recent results from knockout mice implicating multiple physiological roles of aquaporins suggest that the aquaporins may be suitable targets for drug discovery by structure-based and/or high-throughput screening strategies. water transport; protein structure; electron microscopy; crystallography; water permeability