Identification of Nicotinic Acetylcholine Receptor Amino Acids Photolabeled by the Volatile Anesthetic Halothane

Abstract

To identify inhalational anesthetic binding domains in a ligand-gated ion channel, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with [(14)C]halothane and determined by Edman degradation some of the photolabeled amino acids in nAChR subunit fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography. Irradiation at 254 nm for 60 s in the presence of 1 mM [(14)C]halothane resulted in incorporation of approximately 0.5 mol of (14)C/mol of subunit, with photolabeling distributed within the nAChR extracellular and transmembrane domains, primarily at tyrosines. GammaTyr-111 in ACh binding site segment E was labeled, while alphaTyr-93 in segment A was not. Within the transmembrane domain, alphaTyr-213 within alphaM1 and deltaTyr-228 within deltaM1 were photolabeled, while no labeled amino acids were identified within the deltaM2 ion channel domain. Although the efficiency of photolabeling at the subunit level was unaffected by agonist, competitive antagonist, or isoflurane, state-dependent photolabeling was seen in a delta subunit fragment beginning at deltaPhe-206. Labeling of deltaTyr-212 in the extracellular domain was inhibited >90% by d-tubocurarine, whereas addition of either carbamylcholine or isoflurane had no effect. Within M1, the level of photolabeling of deltaTyr-228 with [(14)C]halothane was increased by carbamylcholine (90%) or d-tubocurarine (50%), but it was inhibited by isoflurane (40%). Within the structure of the nAChR transmembrane domain, deltaTyr-228 projects into an extracellular, water accessible pocket formed by amino acids from the deltaM1-deltaM3 alpha-helices. Halothane photolabeling of deltaTyr-228 provides initial evidence that halothane and isoflurane bind within this pocket with occupancy or access increased in the nAChR desensitized state compared to the closed channel state. Halothane binding at this site may contribute to the functional inhibition of nAChRs.

A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor

“…As a mobile contact point between the LBD and TMD, the M2-M3 loop would have an important role in conformational transmission and gating, and this is not the first time that this has been suggested for Cys-loop receptors (67). In the simulation, the loop is seen to curl down into the hydrophobic pocket in the TMD, between M1, M2, and M3 where anesthetics are thought to bind to the receptor (68), and in other Cys-loop receptors such as the GABA receptor (69,70). For nicotinic channels, a view that anesthetics bind solely to M2 (71,72) seems to prevail.…”

Multiple binding sites for the general anesthetic isoflurane identified in the nicotinic acetylcholine receptor transmembrane domain

“…Photolabeling of the nAChR reveals a halothane binding site at the α δ -δ interface (22) and a TDBzl-etomidate binding site at α γ -γ (16), the occupation of which is proposed to positively modulate the nAChR. Moreover, residues in the GABA A and glycine receptors have been identified that contribute to positive modulation of those receptors, using photolabeling and mutagenesis (1,3,(19)(20)(21)(22)(23)(24); some of these residues are thought to lie at the subunit interfaces in a site analogous to the intersubunit sites we observe, according to experimentally confirmed (34) homology models based on the nAChR. A similar site is occupied in simulations of halothane interacting with the nAChR (30,35).…”

“…With embedded cholesterol included, the site is likely too small for occupancy by etomidate or neurosteroids, so it is not surprising that it is not labeled by TDBzl-etomidate (16). A photolabeling study of halothane binding to the nAChR (22) primarily detected labeling of aromatic residues, which are not found in the vicinity of the nAChR intrasubunit sites. A site for ethanol in the center of the subunit of GABA A has been considered in the absence of information regarding the location of various critical residues (19), but the contact residues (Fig.…”

“…At clinical concentrations, most volatile anesthetics positively modulate the GABA A receptor but negatively modulate the nAChR, indicating that some binding sites do not overlap (16). In general, experiments suggest multiple binding sites (17,18) for anesthetics and alcohols on both the nAChR and GABA A receptor: Potential sites have been identified in the TMD, at subunit interfaces (1,3,16,(19)(20)(21)(22)(23)(24) in the nAChR pore, (16,(25)(26)(27)(28)(29), and at various positions in the agonist-binding domain (22). The multitude of potential sites and mechanisms has particularly complicated interpretation of ion current measurements, because of the possibility of competing effects.…”

Gating-enhanced Accessibility of Hydrophobic Sites within the Transmembrane Region of the Nicotinic Acetylcholine Receptor's δ-Subunit

“…Colored cyan are the side chains in the ␦-subunit labeled by TID in an activation-dependent manner (after 10-ms agonist exposure). Also included are the side chains labeled in the desensitized state either by the anesthetic analogs azioctanol and/or azietomidate (green) or by halothane (yellow) (32,33,43). A Connolly surface representation of TID is included to scale.…”