Anesthetic Binding Sites within the Transmembrane Domain of the α7 Nicotinic Acetylcholine Receptor

Abstract

General anesthetics inhibit the α7 neuronal nicotinic acetylcholine receptor (nAChR) with different sensitivities, but the location of anesthetic binding sites remains unclear. Here we used high-resolution solution NMR to study the interaction of general anesthetics ketamine and halothane with the transmembrane domain (TMD) of the human α7 nAChR. The TMD was expressed in E. coli and purified into detergent LDAO micelles. The specific residues involved in anesthetic binding were identified by anesthetics-induced chemical shift changes in 1H-15N HSQC NMR spectra. Ketamine binds to an intrasubunit site involving residues of all four helices at the intracellular end of the TMD. Two halothane interaction sites were detected. The first site was located near the intracellular end of the TMD, similar to the ketamine site, but involved only residues in TM1 and TM2. The second halothane site was formed by residues in the middle of TM1 and TM3. This site was not observed for ketamine binding, probably because ketamine was too large to penetrate deep into the TMD. Interestingly, unlike the previous finding in the α4β2 nAChR, anesthetic binding to the extracellular end of the TMD was not observed in the α7 nAChR. This difference may be responsible for their distinct sensitivity to halothane. In addition, halothane modulated motion less in α7 than in α4β2 on the micro- to milli-second (μs-ms) timescale, as demonstrated by changes in peak intensity, line width, and peak splitting for some residues. This work was supported by NIH grants: R01GM066358, R01GM056257, and R37GM049202.