title: Chemical derivatization and purification of peptide-toxins for probing ion channel complexes.
abstract: Ion channels function as multi-protein complexes made up of ion-conducting α-subunits and regulatory β-subunits. To detect, identify, and quantitate the regulatory β-subunits in functioning K(+) channel complexes, we have chemically derivatized peptide-toxins that specifically react with strategically placed cysteine residues in the channel complex. Two protein labeling approaches have been developed to derivatize the peptide-toxin, charybdotoxin, with hydrophilic and hydrophobic bismaleimides, and other molecular probes. Using these cysteine-reactive peptide-toxins, we have specifically targeted KCNQ1-KCNE1 K(+) channel complexes expressed in both Xenopus oocytes and mammalian cells. The modular design of the reagents should permit this approach to be applied to the many ion channel complexes involved in electrical excitability as well as salt and water homoeostasis.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Zhengmao
authors.initials: Z
authors.lastname: Hua
authors.forename: William R
authors.initials: WR
authors.lastname: Kobertz
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018999:Charybdotoxin / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003545:Cysteine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D051657:KCNQ1 Potassium Channel / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry; D008564:Membrane Potentials; D026902:Potassium Channel Blockers / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013194:Staining and Labeling / Q000379:methods
chemical_list: D051657:KCNQ1 Potassium Channel; D008301:Maleimides; D026902:Potassium Channel Blockers; D024642:Potassium Channels, Voltage-Gated; D018999:Charybdotoxin; D003545:Cysteine