title: On the use of thiol-modifying agents to determine channel topology.
abstract: A powerful tool in the study of cloned ion channels is the combined use of site-directed mutagenesis and chemical modification. Site-directed mutagenesis is used to introduce new cysteine residues at specific positions in a channel protein, and chemical modification by thiol-specific reagents is then used to assess the exposure of the introduced cysteins. This method has been used to assess secondary structure, membrane topology and conformational changes. We report that one commonly used, charged reagent (MTSEA; aminoethyl methanethiosulfonate) can cross the membrane quite readily. We also find that other reagents that are quite membrane-impermeant can cross the membrane when patches are electrically leaky. Both of these undesired effects can be controlled by the use of a thiol scavenger. These findings argue for caution in the use of modifying reagents to determine the membrane topology of channels and other membrane proteins.
authors.affiliation: Department of Neurobiology, Massachusetts General Hospital, Boston 02114, USA.
authors.forename: M
authors.initials: M
authors.lastname: Holmgren
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: G
authors.initials: G
authors.lastname: Yellen
mesh_terms: D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D003545:Cysteine / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009579:Nitrobenzoates; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010539:Permeability; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels; D013438:Sulfhydryl Compounds; D014981:Xenopus
chemical_list: D007202:Indicators and Reagents; D008081:Liposomes; D009579:Nitrobenzoates; D015221:Potassium Channels; D000644:Quaternary Ammonium Compounds; D051637:Shaker Superfamily of Potassium Channels; D013438:Sulfhydryl Compounds; C093171:methanethiosulfonate ethylammonium; C011136:thionitrobenzoic acid; C039795:trimethylethylammonium; D005020:Ethyl Methanesulfonate; D003545:Cysteine