Kinetics of Anesthetic-Induced Conformational Transitions in a Four-α-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 α-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 (Aα 2 -L1M/L38M) 2 , a four-α-helix bundle protein that was designed to model anesthetic binding sites on Cys-loop LGICs. Stopped-flow fluorescence traces obtained upon mixing (Aα 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 (Aα 2 -L1M/L38M) 2 . This was followed by a bi-exponential 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 (Aα 2 -L1M/L38M) 2 . Cyclopropane, an anesthetic which acts with unusually low potency on Cys-loop LGICs, acted with low apparent potency on (Aα 2 -L1M/L38M) 2 . These results suggest that four-α-helix bundle proteins may be useful models of in-vivo sites of action that allow use of a wide range of techniques to better understand how anesthetic binding leads to changes in protein structure and function. KeywordsAnesthesia; Cyclopropane; Desflurane; Fluorescence; Halothane; Isoflurane; Kinetics; Sevoflurane; Spectroscopy; Stopped-flow Inhaled general anesthetics alter the function of numerous proteins (1-5). Although the receptor site(s) responsible for producing anesthesia are not known with certainty, members of an anesthetic-sensitive superfamily of homologous Cys-loop ligand-gated ion channels (LGICs) are believed to be among the most important targets (6-8). Members of this superfamily include the nicotinic acetylcholine, serotonin type 3, γ-aminobutyric acid type A (GABA A ), and † This work was supported by grants from the National Institutes of Health GM61927 and GM58448 for DER and GM55876 and GM65218 for JSJ LGICs are complex molecules that cannot be expressed in significant quantities and at high purity, there remain considerable obstacles to applying powerful biophysical and biochemical approaches to gain insight into how inhaled anesthetics act on these targets.To overcome these obstacles, small, water-soluble four-α-helix bundle proteins have been designed, produced, and used as well-defined model systems to explore how inhaled anesthetics bind to p...

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