title: Kinetics of anesthetic-induced conformational transitions in a four-alpha-helix bundle protein.
abstract: Inhaled anesthetics are thought to alter the conformational states of Cys-loop ligand-gated ion channels (LGICs) by binding within discrete cavities that are lined by portions of four alpha-helical transmembrane domains. Because Cys-loop LGICs are complex molecules that are notoriously difficult to express and purify, scaled-down models have been used to better understand the basic molecular mechanisms of anesthetic action. In this study, stopped-flow fluorescence spectroscopy was used to define the kinetics with which inhaled anesthetics interact with (Aalpha(2)-L1M/L38M)(2), a four-alpha-helix bundle protein that was designed to model anesthetic binding sites on Cys-loop LGICs. Stopped-flow fluorescence traces obtained upon mixing (Aalpha(2)-L1M/L38M)(2) with halothane revealed immediate, fast, and slow components of quenching. The immediate component, which occurred within the mixing time of the spectrofluorimeter, was attributed to direct quenching of tryptophan fluorescence upon halothane binding to (Aalpha(2)-L1M/L38M)(2). This was followed by a biexponential fluorescence decay containing fast and slow components, reflecting anesthetic-induced conformational transitions. Fluorescence traces obtained in studies using sevoflurane, isoflurane, and desflurane, which poorly quench tryptophan fluorescence, did not contain the immediate component. However, these anesthetics did produce the fast and slow components, indicating that they also alter the conformation of (Aalpha(2)-L1M/L38M)(2). Cyclopropane, an anesthetic that acts with unusually low potency on Cys-loop LGICs, acted with low apparent potency on (Aalpha(2)-L1M/L38M)(2). These results suggest that four-alpha-helix bundle proteins may be useful models of in vivo sites of action that allow the use of a wide range of techniques to better understand how anesthetic binding leads to changes in protein structure and function.
authors.affiliation: Department of Anesthesia and Critical Care, Massachusetts General Hospital, 55 Fruit Street, Clinics Building 3, Boston, Massachusetts 02114, USA.
authors.forename: Ken
authors.initials: K
authors.lastname: Solt
authors.forename: Jonas S
authors.initials: JS
authors.lastname: Johansson
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Raines
mesh_terms: D018685:Anesthetics, Inhalation / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D006221:Halothane / Q000737:chemistry*; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008024:Ligands; D008738:Methyl Ethers / Q000737:chemistry*; D008956:Models, Chemical; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D012680:Sensitivity and Specificity; D000077149:Sevoflurane; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D018685:Anesthetics, Inhalation; D007473:Ion Channels; D008024:Ligands; D008738:Methyl Ethers; D011506:Proteins; D000077149:Sevoflurane; D003545:Cysteine; D006221:Halothane