Comprehensive Cysteine-Scanning Mutagenesis Reveals Claudin-2 Pore-Lining Residues with Different Intrapore Locations [Protein Structure and Folding]

January 16th, 2014 by Li, J., Zhuo, M., Pei, L., Rajagopal, M., Yu, A. S. L.

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 MDCK 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 (MTSET) indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal MDCK 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: S68, S47, T62/I66, T56, T32/G45, M52. 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 D65 to S68 that form a major part of the lining of the pore.
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