Claudins are tight junction proteins that determine paracellular permeability and probably form the lining of the paracellular pore. In the renal tubule, they play important roles in electrolyte and acid-base handling. The long-term goal of this proposal is to determine the structural and functional determinants of ionic selectivity in the paracellular pore. We propose to use claudin-2 as a model cation-selective pore. Our overall HYPOTHESIS is that the paracellular cation pore is a relatively wide, water-filled cylinder, the wall of which is formed from the extracellular domains. The side chains of acidic residues within the extracellular domain contribute negatively charged sites that line the pore. We hypothesize that monovalent cations permeate the pore by partially dehydrating and are then stabilized either by discrete electrostatic binding sites, or simply by the altered dielectric environment within the pore.
The SPECIFIC AIMS are to: (1) Characterize the biophysical properties of the claudin-2 paracellular pore and model the permeation of cations through the pore; (2) Identify acidic residues in the extracellular domains of claudin-2 that determine paracellular cation selectivity and characterize their role; (3) Identify other pore-lining residues in claudin-2 by cysteine mutagenesis and determine their location and function METHODS to be used include stable transfection of MDCK I cells with claudin-2, Ussing chamber studies of diffusion potential and conductance, interference with negatively charged residues by pH and carboxyl modifying reagents, site-directed mutagenesis of extracellular residues, and probing of cysteine-substituted residues by methanethiosulfonate reagents. The data obtained will lead to a structural model to explain the function of claudin-based pores. This may shed light on disorders such as hypertension and renal salt-wasting and lead to the development of drugs that could regulate paracellular permeability of the renal tubule and correct electrolyte disorders. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK062283-05A1
Application #
7317040
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
2002-07-01
Project End
2011-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
5
Fiscal Year
2007
Total Cost
$345,202
Indirect Cost
Name
University of Southern California
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Yu, Alan S L (2017) Paracellular transport as a strategy for energy conservation by multicellular organisms? Tissue Barriers 5:e1301852
Yu, Alan S L (2017) Paracellular transport and energy utilization in the renal tubule. Curr Opin Nephrol Hypertens 26:398-404
Rosenthal, R; G├╝nzel, D; Krug, S M et al. (2017) Claudin-2-mediated cation and water transport share a common pore. Acta Physiol (Oxf) 219:521-536
Pei, Lei; Solis, Glenn; Nguyen, Mien T X et al. (2016) Paracellular epithelial sodium transport maximizes energy efficiency in the kidney. J Clin Invest 126:2509-18
Yu, Alan S L (2015) Claudins and the kidney. J Am Soc Nephrol 26:11-9
Weber, Christopher R; Liang, Guo Hua; Wang, Yitang et al. (2015) Claudin-2-dependent paracellular channels are dynamically gated. Elife 4:e09906
Li, Jiahua; Zhuo, Min; Pei, Lei et al. (2014) Comprehensive cysteine-scanning mutagenesis reveals Claudin-2 pore-lining residues with different intrapore locations. J Biol Chem 289:6475-84
Gunzel, Dorothee; Yu, Alan S L (2013) Claudins and the modulation of tight junction permeability. Physiol Rev 93:525-69
Li, Jiahua; Zhuo, Min; Pei, Lei et al. (2013) Conserved aromatic residue confers cation selectivity in claudin-2 and claudin-10b. J Biol Chem 288:22790-7
Li, Jiahua; Angelow, Susanne; Linge, Anna et al. (2013) Claudin-2 pore function requires an intramolecular disulfide bond between two conserved extracellular cysteines. Am J Physiol Cell Physiol 305:C190-6

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