PMID: 8938712Jan 1, 1996Paper

On the use of thiol-modifying agents to determine channel topology

Neuropharmacology
M HolmgrenG Yellen

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.

References

Sep 1, 1991·Biophysical Journal·G F TomaselliG Yellen
Sep 1, 1996·The Journal of General Physiology·M HolmgrenG Yellen

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Citations

Feb 7, 2012·Biochemistry·Ming-Feng TsaiChristopher Miller
Jul 21, 2011·Nature Communications·Tianbo LiCecilia M Canessa
Jul 1, 2008·Nature Neuroscience·Mufeng LiKenton J Swartz
Jun 30, 2009·Nature Structural & Molecular Biology·Héctor SalazarTamara Rosenbaum
Jul 24, 2008·Proceedings of the National Academy of Sciences of the United States of America·Lucy R ForrestGary Rudnick
Mar 27, 2013·Proceedings of the National Academy of Sciences of the United States of America·Ming-Feng Tsai, Christopher Miller
Mar 27, 2013·Proceedings of the National Academy of Sciences of the United States of America·Ming-Feng TsaiChristopher Miller
Dec 29, 2009·The Journal of Biological Chemistry·Cassandra S Arendt, Buddy Ullman
Sep 16, 2011·The Journal of Biological Chemistry·Qian ShiJean X Jiang
Mar 1, 2012·The Journal of Biological Chemistry·Marcelo D Carattino, Margaret C Della Vecchia
Apr 15, 2008·The Journal of Biological Chemistry·Chunlong MaLawrence H Pinto
Jan 23, 2009·The Journal of Biological Chemistry·Quansheng ZhuIra Kurtz
Sep 25, 1999·The Journal of General Physiology·L HeginbothamC Miller
Feb 2, 2000·The Journal of General Physiology·S K Tiwari-WoodruffD M Papazian
Oct 5, 2001·The Journal of General Physiology·S S SmithD C Dawson
Jun 27, 2002·The Journal of General Physiology·Manuel SimoesRémy Sauvé
Dec 17, 2003·The Journal of General Physiology·Sriharsha VemanaH Peter Larsson
Oct 13, 2010·The Journal of General Physiology·Junjie Wang, Gerhard Dahl
Jun 1, 2011·The Journal of General Physiology·Sara I Börjesson, Fredrik Elinder
Jun 1, 1997·The EMBO Journal·F RassendrenA Surprenant
Jan 7, 2010·The Journal of Pharmacology and Experimental Therapeutics·Hacer S G BüyüknacarSerpil Onder
Jun 10, 2005·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Yolanda F VallejoWilliam N Green
Aug 5, 1998·Proceedings of the National Academy of Sciences of the United States of America·J V Ferrer, J A Javitch

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