Combining Mutations and Electrophysiology to Map Anesthetic Sites on Ligand-Gated Ion Channels.
General anesthetics are known to act in part by binding to and altering the function of pentameric ligand-gated ion channels such as nicotinic acetylcholine and γ-aminobutyric acid type A receptors. Combining heterologous expression of the subunits that assemble to form these ion channels, mutagenesis techniques and voltage-clamp electrophysiology have enabled a variety of "structure-function" approaches to questions of where anesthetic binds to these ion channels and how they enhance or inhibit channel function. Here, we review the evolution of concepts and experimental strategies during the last three decades, since molecular biological and electrophysiological tools became widely used. Topics covered include: (1) structural models as interpretive frameworks, (2) various electrophysiological approaches and their limitations, (3) Monod-Wyman-Changeux allosteric models as functional frameworks, (4) structural strategies including chimeras and point mutations, and (5) methods based on cysteine substitution and covalent modification. We discuss in particular depth the experimental design considerations for substituted cysteine modification-protection studies.
['Forman|Stuart A|SA|']
D000494:Allosteric Regulation D000777:Anesthetics D000818:Animals D001665:Binding Sites D003545:Cysteine D004594:Electrophysiology D057809:HEK293 Cells D006801:Humans D015640:Ion Channel Gating D051379:Mice D016297:Mutagenesis, Site-Directed D009865:Oocytes D018408:Patch-Clamp Techniques D017354:Point Mutation D000072417:Protein Domains D011963:Receptors, GABA-A D014982:Xenopus laevis D064569:alpha7 Nicotinic Acetylcholine Receptor
General anesthetic Cysteine Covalent modification GABA(A) receptor Nicotinic acetylcholine receptor Xenopus oocyte