Anesthetic Binding Sites in the Neuronal N-Acetylcholine Receptor Transmembrane Domain

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to general anesthetics at physiologically relevant concentrations, but the locations and mechanisms of relevant anesthetic interactions are unknown. The interactions of the anesthetics halothane, isoflurane, and ketamine with the transmembrane domains of the human nAChR α4 and β2 subunits were studied using fluorescence spectroscopy and high-resolution solution NMR. The isolated transmembrane domains, with the extracellular and intracellular domains removed by mutagenesis, were expressed in E. coli and purified into detergent micelles. Multi-angle light scattering experiments on a mixture of α4 and β2 subunits demonstrated their interaction in detergent micelles. Anesthetics quenched the intrinsic tryptophan fluorescence of these proteins in a manner consistent with specific binding. Direct interaction with anesthetic halothane, isoflurane, and ketamine was confirmed by NMR saturation transfer difference spectroscopy. The anesthetics induced chemical shift changes for specific resonances in the NMR spectra of the α4 and β2 transmembrane domains. Two specific interaction sites for the volatile anesthetics halothane and isoflurane have been identified at the extracellular and intracellular interfaces near the ends of the transmembrane domain in the α4 subunit. Differing from halothane and isoflurane, the intravenous anesthetic ketamine affected a set of residues at a more interior location near the extracellular interface. Changes in NMR peak intensity and line width indicated modulations of protein motion by all three anesthetics. This study demonstrated that general anesthetics interact at specific sites within the isolated transmembrane domain of nAChR and affect protein dynamics on a time scale consistent with protein function. This work was supported by NIH grants: R01GM066358, R01GM056257, R37GM049202, and R01GM069766.