Kinetics of anesthetic-induced conformational transitions in a four-alpha-helix bundle protein.

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 (GABAA), and glycine receptors (9, 10). Electrophysiological studies using receptor chimeras and site-directed mutagenesis techniques suggest that inhaled anesthetics bind to LGICs within discrete protein cavities that are lined by portions of four α-helical transmembrane domains (M1-M4), producing conformational states with increased sensitivity to agonist (11-14). In the case of the GABAA receptor, inhaled anesthetics can also directly activate the ion channel even in the absence of agonist (15, 16). Both of these actions may occur if general anesthetics stabilize receptors in an open channel state. However because Cys-loop 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 proteins (17-21). Each four-α-helix bundle protein is a dimer of two 27-residue α-helices joined by an eight-residue glycine linker (figure 1). It contains a hydrophobic cavity that is lined by the four α-helices and binds inhaled anesthetics. Recently, it has been shown that such binding can alter the protein's conformational state as evidenced by changes in its fluorescence, circular dichroism and nuclear magnetic resonance spectra following equilibration with anesthetic (22). Such observations suggest that four-α-helix bundle proteins can serve not only as models to explore how anesthetics bind, but also how such binding leads to changes in protein structure and function. Figure 1 (A) Modeled structure of the expressed four-α-helix bundle (Aα2-L1M/L38M)2. The cylinders represent the two 27-residue amphiphilic α-helical portions of each 62-residue di-α-helical peptide, joined by an eight-residue glycine ... The present study was undertaken to examine the kinetics with which inhaled anesthetics interact with the four-α-helix bundle protein (Aα2-L1M/L38M)2. Stopped-flow fluorescence spectroscopic techniques having millisecond time resolution were used to monitor anesthetic-protein interactions. This was possible because each peptide in (Aα2-L1M/L38M)2 contains a tryptophan residue at position 15 (W15) whose fluorescence is reduced upon equilibration with clinically relevant concentrations of anesthetic (22, 23). We focused our studies on two inhaled anesthetics, halothane and sevoflurane. Halothane directly and efficiently quenches tryptophan fluorescence (24) whereas sevoflurane does not (22).