Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.
(17/469)
Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene. (+info)
Inhibition of aquaporin-1 water permeability by tetraethylammonium: involvement of the loop E pore region.
(18/469)
Previously, the only known blockers of water permeability through aquaporin-1 (AQP1) water channels were mercurial reagents such as HgCl(2). For AQP1, inhibition by mercury has been attributed to the formation of a mercaptide bond with cysteine residue 189 found in the putative pore-forming region loop E. Here we show that the nonmercurial compound, tetraethylammonium (TEA) chloride, reduces the water permeability of human AQP1 channels expressed in Xenopus oocytes. After preincubation of the oocytes for 15 min with 100 microM TEA, AQP1 water permeability was reduced by 20 to 40%, a degree of partial block similar to that obtained with 15 min of incubation in 100 microM HgCl(2). The reduction of water permeability was dose-dependent for tested concentrations up to 10 mM TEA. TEA blocks the Shaker potassium channel by interacting with a tyrosine residue in the outer pore region. We tested whether an analogous tyrosine residue in loop E of AQP1 could be involved in the binding of TEA. Using polymerase chain reaction, tyrosine 186 in AQP1, selected for its proximity to the mercury-binding site, was mutated to phenylalanine (Y186F), alanine (Y186A), or asparagine (Y186N). Oocyte expression of the mutant AQP1 channels showed that the water permeability of Y186F was equivalent to that of wild-type AQP1; the other mutant channels did not conduct water. However, in contrast to wild-type AQP1, the water permeability of Y186F was not reduced with 100 microM TEA. These results suggest that TEA reduces AQP1 water permeability by interacting with loop E. (+info)
Luminal hypotonicity in proximal tubules of aquaporin-1-knockout mice.
(19/469)
To examine the role of aquaporin-1 (AQP1) in near-isosmolar fluid reabsorption in the proximal tubule, we compared osmolalities in micropuncture samples of late proximal tubular fluid and plasma in wild-type (+/+) and AQP1-knockout (-/-) mice. Compared with matched wild-type mice, the -/- animals produce a relatively hypotonic urine (607 +/- 42 vs. 1,856 +/- 101 mosmol/kgH(2)O) and have a higher plasma osmolality under micropuncture conditions (346 +/- 11 vs. 318 +/- 5 mosmol/kgH(2)O; P < 0.05). Measurements of tubular fluid osmolality were done in three groups of mice, +/+, -/-, and hydrated -/- mice in which plasma osmolality was reduced to 323 +/- 1 mosmol/kgH(2)O. Late proximal tubular fluid osmolalities were 309 +/- 5 (+/+, n = 21), 309 +/- 4 (-/-, n = 24), and 284 +/- 3 mosmol/kgH(2)O (hydrated -/-, n = 19). Tubular fluid chloride concentration averaged 152 +/- 1 (+/+), 154 +/- 1 (-/-), and 140 +/- 1 mM (hydrated -/-). Transtubular osmotic gradients in untreated and hydrated AQP1 -/- mice were 39 +/- 4 (n = 25) and 39 +/- 3 mosmol/kgH(2)O (n = 19), values significantly higher than in +/+ mice (12 +/- 2 mosmol/kgH(2)O; n = 24; both P < 0.001). AQP1 deficiency in mice generates marked luminal hypotonicity in proximal tubules, resulting from the retrieval of a hypertonic absorbate and indicating that near-isosmolar fluid absorption requires functional AQP1. (+info)
Role of aquaporins in alveolar fluid clearance in neonatal and adult lung, and in oedema formation following acute lung injury: studies in transgenic aquaporin null mice.
(20/469)
Aquaporin (AQP) water channels provide a major pathway for osmotically driven water movement across epithelial and microvascular barriers in the lung. We used mice deficient in each of the three principal lung aquaporins, AQP1, AQP4 and AQP5, to test the hypothesis that aquaporins are important in neonatal lung fluid balance, adult lung fluid clearance and formation of lung oedema after acute lung injury. Wet-to-dry weight ratios (W/D) in lungs from wild-type mice decreased from 7.9 to 5.7 over the first hour after spontaneous delivery. AQP deletion did not significantly affect W/D at 45 min after birth. Alveolar fluid clearance was measured in living ventilated mice in which 0.5 ml saline containing radiolabelled albumin was instilled into the airspaces. Fluid clearance was 17.4 % in 15 min and inhibited >90 % by amiloride, but clearance was not affected by AQP deletion. W/D was measured in established models of acute lung injury - acid aspiration and thiourea administration. Two hours after intratracheal administration of HCl, W/D increased from 3.7 to 7.5 but was not affected by AQP deletion. Three hours after intraperitoneal infusion of thiourea, W/D increased to 5.5 and marked pleural effusions appeared, but there were no differences in wild-type and AQP knockout mice. Hyperoxic subacute lung injury was induced by 95 % oxygen. Neither mean survival (143 h) nor W/D at 65 h (5.1) were significantly affected by AQP deletion. Despite their role in osmotically driven lung water transport, aquaporins are not required for the physiological clearance of lung water in the neonatal or adult lung, or for the accumulation of extravascular lung water in the injured lung. (+info)
Dysregulation of renal aquaporins and Na-Cl cotransporter in CCl4-induced cirrhosis.
(21/469)
BACKGROUND: Severe hepatic cirrhosis is associated with abnormal renal water retention. METHODS: Semiquantitative immunoblotting was employed to investigate the abundance of the major renal aquaporins (water channels) and sodium-dependent cotransporters in kidneys from control rats and rats with cirrhosis secondary to chronic CCl4 inhalation. RESULTS: The cirrhotic rats had ascites and manifested a water excretion defect detected by a standard water-loading test. The abundance of aquaporin-1 (the major aquaporin in the proximal tubule) was increased, an effect markedly accentuated in high-density membrane fractions prepared by differential centrifugation. Differential centrifugation studies demonstrated a redistribution of aquaporin-2 from high-density to low-density membranes, compatible with increased trafficking of aquaporin-2 to the plasma membrane. The abundance of aquaporin-3, but not aquaporin-2, was increased in collecting ducts of rats with CCl4-induced cirrhosis. The Na-K-2Cl cotransporter of the thick ascending limb showed no change in abundance. However, the abundance of the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule was markedly suppressed in cirrhotic rats, possibly contributing to a defect in urinary dilution. CONCLUSIONS: In this model of cirrhosis, the development of a defect in urinary dilution may be multifactorial, with contributions from at least four abnormalities in transporter regulation: (1) an increase in the renal abundance of aquaporin-1, (2) a cellular redistribution of aquaporin-2 in the collecting duct compatible with trafficking to the plasma membrane without an increase in total cellular aquaporin-2, (3) an increase in the renal abundance of aquaporin-3, and (4) a decrease in the abundance of the thiazide-sensitive cotransporter of the distal convoluted tubule. (+info)
Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria.
(22/469)
BACKGROUND: Cisplatin (CP)-induced polyuria in rats is attributed to decreased medullary hypertonicity and/or an end-organ resistance to vasopressin. However, the roles of renal aquaporins (AQPs) have not yet been explored. METHODS: Male Sprague-Dawley rats (230 to 245 g) received either a single injection of CP (5 mg/kg, N = 4) or saline (N = 4) intraperitoneally five days before sacrifice. Urine, blood, and kidney samples were analyzed. RESULTS: Platinum accumulated in the cortex and outer medulla of CP-treated rats (39.05 +/- 7.50 and 36.48 +/- 12.44 microg/g vs. 2.52 +/- 0.43 and 1.87 +/- 0.84 microg/g dry tissue in controls, respectively). Histologically, tubular damage and decreased AQP1 immunolabeling were detected in the S3 segment of proximal tubules. CP treatment caused 4.4- and 4.8-fold increases, respectively, in blood urea nitrogen and urine volume, and a 4. 4-fold decrease in urine osmolality. Immunoblots showed that AQP2 and AQP3 were significantly reduced to 33 +/- 10% (P < 0.001) and 69 +/- 11% (P < 0.05), respectively, in the inner medulla of CP-treated rats. Immunocytochemical analysis showed a decrease in AQP2 labeling in the inner medulla of CP-treated rats. Northern hybridization revealed a 33 +/- 11% (P < 0.002) decrease in AQP2 mRNA expression in the inner medulla of CP-treated rats. AQP1 protein expression levels were modestly (67 +/- 7%, P = 0.057) and significantly (53 +/- 13%, P < 0.007) decreased in outer and inner medullae, respectively, of CP-treated rats. CONCLUSIONS: CP-induced polyuria in rats is associated with a significant decrease in the expression of collecting duct (AQP2 and AQP3) and proximal nephron and microvascular (AQP1) water channels in the inner medulla. (+info)
Mesoscopic surfactant organization and membrane protein crystallization.
(23/469)
The paucity of detailed X-ray crystallographic structures of integral membrane proteins arises from substantive technical obstacles in the overexpression of multimilligram quantities of protein, and in the crystallization of purified protein-detergent complexes (PDCs). With rare exception, crystal contacts within the lattice are mediated by protein-protein interaction, and the detergent surrounding the protein behaves as a disordered solvent. The addition and use of surfactants that display mesoscopic self-assembly behavior in membrane protein crystallization experiments presents a novel alternative strategy. Well-ordered crystals of the water channel human aquaporin-1 (hAQP1) that diffract to 4 A resolution have been obtained with this approach. (+info)
Identification of sequence determinants that direct different intracellular folding pathways for aquaporin-1 and aquaporin-4.
(24/469)
Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function. (+info)