The long term goal of the proposed studies remains the definition of water transport mechanisms across biological membranes at the physiological, biochemical and molecular levels. In this revised proposal, efforts will be focused on the identification and structure-function analysis of biologically important water transporting proteins.
Specific Aim 1 : To identify and characterize novel mammalian water transporters. Based on the tissue distribution of water channels identified thus far, additional physiologically important water channels must exist. A PcR/homology cloning strategy has already yielded 3 new full-length and 3 partial-length cDNAs. We propose: a. to obtain full length cDNAs corresponding to the putative new water channels, b. characterize protein function by expression in Xenopus oocytes, and c. determine tissue distribution by Northern blot, in situ hybridization and immunostaining.
Specific Aim 2 : To quantify the biophysical transporting properties of water channels. The key functional parameters that characterize water channel proteins will be measured, including: single channel water permeability, solute selectivity, osmotic-to-diffusional water permeability ratio, and Arrhenius activation energy. In addition, putative inhibitory and activating compounds will be tested. Experiments will be carried out on cloned proteins CHIP28, WCH-CD, MIWC, GLIP, and the new water channels identified in aim 1. Novel aspects of this aim include measurement of osmotic and diffusional water permeability by new fluorescence methods, and liposome reconstitution of proteins purified from a baculovirus/Sf9 expression system.
Specific Aim 3 : To study selected features of water channel structure. Initial experiments indicated that CHIP28 is assembled in membranes as tetramers in which individual monomers function independently and contain multiple membrane-spanning helical domains. There are three subaims: a. To determine whether WCH-CD and MIWC form oligomers in membranes by freeze fracture electron microscopy and electron crystallography. b. To determine the transmembrane topology of MIWC by translation of selected mutated, chimeric and flagged cDNAs. We will also test the provocative hypothesis that functional maturation of CHIP28 occurs between the endoplasmic reticulum and plasma membrane and is associated with a change in topology. c. Fluorescence energy transfer and time-resolved fluorescence will be applied to measure selected distances between labeled residues in CHIP28 and to test whether HgCl2 inhibition influences protein segmental dynamics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK035124-12
Application #
2684146
Study Section
General Medicine B Study Section (GMB)
Project Start
1986-01-01
Project End
1999-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
12
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Smith, Alex J; Verkman, Alan S (2018) The ""glymphatic"" mechanism for solute clearance in Alzheimer's disease: game changer or unproven speculation? FASEB J 32:543-551
Tradtrantip, Lukmanee; Felix, Christian M; Spirig, Rolf et al. (2018) Recombinant IgG1 Fc hexamers block cytotoxicity and pathological changes in experimental in vitro and rat models of neuromyelitis optica. Neuropharmacology 133:345-353
Agbani, Ejaife O; Williams, Christopher M; Li, Yong et al. (2018) Aquaporin-1 regulates platelet procoagulant membrane dynamics and in vivo thrombosis. JCI Insight 3:
Verkman, Alan S; Smith, Alex J; Phuan, Puay-Wah et al. (2017) The aquaporin-4 water channel as a potential drug target in neurological disorders. Expert Opin Ther Targets 21:1161-1170
Yao, Xiaoming; Verkman, Alan S (2017) Marked central nervous system pathology in CD59 knockout rats following passive transfer of Neuromyelitis optica immunoglobulin G. Acta Neuropathol Commun 5:15
Lee, Sujin; Phuan, Puay-Wah; Felix, Christian M et al. (2017) Nanomolar-Potency Aminophenyl-1,3,5-triazine Activators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel for Prosecretory Therapy of Dry Eye Diseases. J Med Chem 60:1210-1218
Verkman, Alan S; Tradtrantip, Lukmanee; Smith, Alex J et al. (2017) Aquaporin Water Channels and Hydrocephalus. Pediatr Neurosurg 52:409-416
Cil, Onur; Phuan, Puay-Wah; Gillespie, Anne Marie et al. (2017) Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J 31:751-760
Smith, Alex J; Yao, Xiaoming; Dix, James A et al. (2017) Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma. Elife 6:
Tradtrantip, Lukmanee; Yao, Xiaoming; Su, Tao et al. (2017) Bystander mechanism for complement-initiated early oligodendrocyte injury in neuromyelitis optica. Acta Neuropathol 134:35-44

Showing the most recent 10 out of 436 publications