Cysteine mutagenesis to study the structure of claudin-2 paracellular pores.
The structure and transport mechanism of paracellular pores are only poorly understood. Here we describe for the first time how the substituted cysteine accessibility method (SCAM), previously developed to study transmembrane transport, can be applied to analyze the pathway of paracellular ion permeation. Using stable transfected Madin Darby canine kidney type I cells, induced to express claudin-2, we show that paracellular cation transport can be blocked by sulfhydryl-specific methanethiosulfonate (MTS) and that SCAM can be used to identify residues that line paracellular pores.
Angelow
Susanne
S
Department of Medicine, Division of Nephrology, University of Southern California Keck School of Medicine, Los Angeles, California 90089, USA. angelow@usc.edu
Yu
Alan S L
AS
D000818:Animals; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis
D008565:Membrane Proteins; D003545:Cysteine