Time-Resolved Photolabeling of the Nicotinic Acetylcholine Receptor by [3H]Azietomidate, an Open-State Inhibitor

General anesthetics of diverse structures, including volatile and intravenous anesthetics and alcohols, modulate the function of members of the “Cys-loop” superfamily of neurotransmitter-gated ion channels either by potentiating agonist responses at inhibitory GABAA receptors and glycine receptors or by inhibiting responses of the excitatory serotonin 5-HT3 and nicotinic acetylcholine receptors (nAChRs) (Yamakura et al., 2001; Arias et al., 2003; Hemmings et al., 2005). Each Cys-loop receptor is a pentamer of homologous or identical subunits that associate at a central axis that is the ion channel. Structural models can be derived from the structure of the muscle-type nAChR based upon cryoelectron microscope images of the Torpedo marmorata nAChR (Unwin, 2005), which provides a definition of the tertiary structure of the nAChR but lacks the resolution necessary to reliably define the positions of individual amino acids. The N-terminal half of each subunit contributes to the extracellular domain, containing the neurotransmitter binding sites that are located at subunit interfaces (α-γ and α-δ in the α2βγδ Torpedo californica nAChR) 30 A above the level of the membrane. Each nAChR subunit's transmembrane domain is made up of a loose bundle of four α helices (M1-M4), with amino acids from each M2 helix contributing to the lumen of the ion channel and M4 located most peripherally and in greatest contact with lipid. The structure of nAChR transmembrane domain suggests the existence of pockets within each subunit helix bundle and at subunit interfaces that are potential binding sites for general anesthetics and other allosteric modulators. In the absence of agonist, the nAChR exists predominantly in a resting, closed channel conformation with low affinity for agonist that is in equilibrium with a nonconducting desensitized state with highest affinity for agonist. Agonist binding induces conformational transitions, within a fraction of a millisecond, to an open channel state (Maconochie and Steinbach, 1998), and desensitization occurs in two kinetically distinct phases over times from 0.1 to 100 s (Hess et al., 1983). Mutational analyses identified positions within the M2 helices that can enhance or reduce the potency of aliphatic alcohols as open-state inhibitors (Zhou et al., 2000; Wenningmann et al., 2001; Borghese et al., 2002). Because these mutations often affect ACh gating in the absence of anesthetics, and they include substitutions at positions oriented toward the lumen of the ion channel and toward the interior of each subunit helix bundle or the interfaces with another subunit, it is difficult to determine whether the positions contribute directly to anesthetic binding or are involved in allosteric modulation of gating. Photoaffinity labeling provides a method to directly identify amino acids contributing to a drug binding site in a protein without any prior assumptions about the location of the site (Kotzyba-Hibert et al., 1995; Vodovozova, 2007). State-dependent drug binding sites have been mapped by photolabeling T. californica nAChRs in the absence of agonist (resting state), equilibrated with agonist (desensitized state), and by the use of rapid-mixing and freeze-quench techniques (Arevalo et al., 2005), in the conformations transiently stabilized by agonist (for review, see Mourot et al., 2006). R(+)-Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and as a potentiator of the responses to submaximal concentrations of GABA, whereas at concentrations above 10 μM, it inhibits nAChRs. Azietomidate is a photoreactive etomidate analog that is equipotent with etomidate as a general anesthetic and as a positive allosteric modulator of GABAARs (Husain et al., 2003; Liao et al., 2005). Photolabeling studies with [3H]azietomidate identified a binding site in the GABAAR transmembrane domain at the interface between the β and α subunits (Li et al., 2006). In the T. californica nAChR, azietomidate is equipotent with etomidate as a noncompetitive antagonist, binding with 10-fold higher affinity to the nAChR in the desensitized state than the resting state and photolabeling amino acids near the extracellular end of the ion channel domain only in the desensitized state (position M2-M20: αGlu-262 and δGln-276) (Ziebell et al., 2004). However, it is not known where (or whether) it interacts with nAChRs in the open channel state. To provide a further definition of the diversity of binding sites and state dependence of binding of a single general anesthetic in a “Cys-loop” receptor, we use rapid perfusion electrophysiological techniques to characterize azietomidate's state dependence and kinetics of inhibition, and we use time-resolved photolabeling to identify the nAChR binding sites occupied after exposure to [3H]azietomidate and agonist for 50 ms (open state) compared with the desensitized state.