title: Comprehensive cysteine-scanning mutagenesis reveals Claudin-2 pore-lining residues with different intrapore locations.
abstract: The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. We aimed to map the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutations within the ECL1 by expression in polyclonal Madin-Darby canine kidney II Tet-Off cells and found nine mutants that displayed a significant decrease of conductance after treatment with the thiol-reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate, indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal Madin-Darby canine kidney I Tet-Off cells and exposed them to thiol-reactive reagents. The maximum degree of inhibition of conductance, size selectivity of degree of inhibition, and size dependence of the kinetics of reaction were used to deduce the location of residues within the pore. Our data support the following sequence of pore-lining residues located from the narrowest to the widest part of the pore: Ser(68), Ser(47), Thr(62)/Ile(66), Thr(56), Thr(32)/Gly(45), and Met(52). The paracellular pore appears to primarily be lined by polar side chains, as expected for a predominantly aqueous environment. Furthermore, our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp(65)-Ser(68) that form a major part of the lining of the pore.
authors.affiliation: Division of Nephrology and Hypertension and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160; Department of Medicine, John H. Stroger, Jr. Hospital of Cook County, Chicago, Illinois 60612.
authors.forename: Jiahua
authors.initials: J
authors.lastname: Li
authors.affiliation: Division of Nephrology and Hypertension and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160.
authors.forename: Min
authors.initials: M
authors.lastname: Zhuo
authors.affiliation: Division of Nephrology and Hypertension and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160.
authors.forename: Lei
authors.initials: L
authors.lastname: Pei
authors.affiliation: Division of Nephrology and Hypertension and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160.
authors.forename: Madhumitha
authors.initials: M
authors.lastname: Rajagopal
authors.affiliation: Division of Nephrology and Hypertension and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160. Electronic address: ayu@kumc.edu.
authors.forename: Alan S L
authors.initials: ASL
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057167:Claudins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004848:Epithelium / Q000378:metabolism* / Q000648:ultrastructure; D005820:Genetic Testing; D061985:Madin Darby Canine Kidney Cells; D008698:Mesylates / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016062:Porosity; D017433:Protein Structure, Secondary
chemical_list: D057167:Claudins; C113371:Cldn2 protein, mouse; D008698:Mesylates; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine
keywords: Epithelial Cell; Ion Channels; Membrane Transport; Sodium Transport; Tight Junctions