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.
Highlights
The composition of the claudin paracellular pore region is incompletely known
Our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp65–Ser68 that form a major part of the lining of the pore
Characterization of Polyclonal MDCK II Tet-Off Cell Lines Expressing Each Cysteine Mutant—We generated polyclonal, inducible MDCK II Tet-Off cell lines expressing each of the 45 cysteine mutants of the claudin-2 ECL1 by retroviral transduction
Summary
Results: Cysteine-scanning mutagenesis of the first extracellular domain of claudin-2 was used to identify all pore-lining residues and their intrapore locations. The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. All claudins have a large first extracellular loop (ECL1) that is believed to form the lining of paracellular ion pores [3, 4], probably by the interaction of two claudin molecules on adjacent cells (in trans), as well as the interaction of two or more neighboring molecules within the same cell (in cis). The structure and molecular mechanism of claudin-2 has been well studied It forms high conductance, paracellular cation-selective pores (6 – 8). In this study we set out to map out all the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of the entire ECL1. The goal was to provide new insights into the structure-function relationships of the claudin-2 pore, which might be generalizable to other pore-forming claudins
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