Abstract

Numerous physiological and pathophysiological processes involve the transport of water across cell membranes. The molecular identity of water transporters became known with discovery of the aquaporin family of membrane water channels. Four of the ten known mammalian aquaporins are expressed in anterior segment of eye. This application addresses the molecular mechanisms regulating function of human AQPO, AQP1, AQP3, and AQP5 and dysfunction of these proteins in clinical disorders of eye.
Aim I. Analysis of aquaporin proteins in normal human eye. New reagents will be prepared including plasmids encoding wild-type, site-directed mutant, and epitope-tagged human AQPO, AQP1, AQP3, and AQP5. Antibodies specific for the N- and C-termini of the human proteins will be raised in rabbits and affinity-purified. The biophysical functions of human aquaporins will be expressed in Xenopus laevis oocytes and analyzed at baseline and after activation. Human aquaporins will be expressed in yeast and purified for reconstitution into proteoliposomes for permeation studies, into planar bilayers for analysis of electrophysiological properties, and into membrane crystals for structural studies. The distribution of these aquaporins will be defined in normal human eye.
Aim II. Analysis of aquaporin proteins in clinical disorders of eye. The distribution of human aquaporins will be defined in tissues from patients with cataract or Sjogren's syndrome. Basic mechanisms by which AQPO and AQP5 may contribute to these disorders will be sought including defects in water and solute permeation, membrane trafficking, subunit oligomerization, and internalization, Physiological deficits will also be evaluated in rodent models of AQPO degradation and AQP5 deficiency.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY011239-05
Application #
6383997
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Fisher, Richard S
Project Start
1996-02-01
Project End
2006-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
5
Fiscal Year
2001
Total Cost
$367,875
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Rash, J E (2010) Molecular disruptions of the panglial syncytium block potassium siphoning and axonal saltatory conduction: pertinence to neuromyelitis optica and other demyelinating diseases of the central nervous system. Neuroscience 168:982-1008
Huang, Chunyi George; Lamitina, Todd; Agre, Peter et al. (2007) Functional analysis of the aquaporin gene family in Caenorhabditis elegans. Am J Physiol Cell Physiol 292:C1867-73
Liu, Kun; Nagase, Hiroaki; Huang, Chunyi George et al. (2006) Purification and functional characterization of aquaporin-8. Biol Cell 98:153-61
Huang, Chunyi George; Agre, Peter; Strange, Kevin et al. (2006) Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening. Mol Biotechnol 32:83-6
Beitz, Eric; Liu, Kun; Ikeda, Masahiro et al. (2006) Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells. Biol Cell 98:101-9
Eid, Tore; Lee, Tih-Shih W; Thomas, Marion J et al. (2005) Loss of perivascular aquaporin 4 may underlie deficient water and K+ homeostasis in the human epileptogenic hippocampus. Proc Natl Acad Sci U S A 102:1193-8
Liu, Kun; Kozono, David; Kato, Yasuhiro et al. (2005) Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution. Proc Natl Acad Sci U S A 102:2192-7
Liu, Zijuan; Carbrey, Jennifer M; Agre, Peter et al. (2004) Arsenic trioxide uptake by human and rat aquaglyceroporins. Biochem Biophys Res Commun 316:1178-85
Ikeda, Masahiro; Beitz, Eric; Kozono, David et al. (2002) Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63. J Biol Chem 277:39873-9
Liu, Zijuan; Shen, Jian; Carbrey, Jennifer M et al. (2002) Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9. Proc Natl Acad Sci U S A 99:6053-8

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