Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor

Abstract

The excitatory nicotinic acetylcholine receptors (nAChRs) 2 and serotonin 5-HT 3 receptors and the inhibitory ␥-aminobutyric acid type A receptors (GABA A Rs) and glycine receptors are members of the Cys-loop superfamily of neurotransmittergated ion channels (1-3). Models of the three-dimensional structures of these receptors can be derived from the cryoelectron microscopy structure of the Torpedo nAChR in the absence of agonist (4) and from the high resolution crystal structures of distantly related prokaryotic channels (5, 6) and the soluble molluscan acetylcholine-binding proteins (for review, see Ref. 7).The Torpedo nAChR is composed of four homologous subunits (␣ 2 ␤␥␦) that assemble around an axis perpendicular to the membrane, forming a cation-conducting channel. Each subunit is made up of three structural domains; that is, an N-terminal extracellular domain, with the two ACh binding sites at the interfaces between the ␣-␥ and ␣-␦ subunits (8), a transmembrane domain made up of a loose bundle of four transmembrane helices (M1-M4), and a cytoplasmic domain between the M3 and M4 helices (M3-M4 loop). Amino acids on one face of each M2 helix form the lining of the ion channel, and the M1, M3, and M4 helices form an outer ring that is in contact with membrane lipids (4, 9).Muscle or neuronal nAChRs are targets for drugs potentially useful for treatment of myasthenia gravis and CNS disorders, including nicotine addiction, Alzheimer disease, and schizophrenia (10). In an attempt to develop drugs with strong selectivity for subtypes of muscle or neuronal nAChRs, screens of natural products and synthetic libraries have identified many drugs that act either as positive or negative allosteric modulators (11,12). However, there is limited information about the diversity of allosteric modulator binding sites or whether positive and negative modulators can bind to the same site, as occurs, for example, at the benzodiazepine site in the GABA A R (13).In addition to binding sites for non-competitive antagonists within the ion channel (14), the transmembrane domain con-

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

“…Our results establish that dFBr can photoincorporate into a wide range of amino acid side chains; however, we did not find photolabeling of amino acids in the TMD that contribute to the known intersubunit or intrasubunit binding sites for Torpedo nAChR positive and negative allosteric modulators (Nirthanan et al, 2008;Hamouda et al, 2011Hamouda et al, , 2014Jayakar Fig. 10.…”

“…Centrifugation assays (Hamouda et al, 2011) H]PCP (1Carb, 6300 mM proadifen, 9000 6 80/1310 6 15 cpm; 1a-BgTx, 6300 mM tetracaine, 3300 6 90/1520 6 15 cpm); and for [ 3 H]tetracaine (1 a-BgTx, 6 300 mM tetracaine, 23,600 6 1800/4360 6 35 cpm). For direct [ using eqs.…”

“…(Garcia et al, 2007;Hamouda et al, 2011). In initial analytical photolabeling experiments, [ 3 H]dFBr photoincorporation into the nAChR was compared after photolysis at 254 nm (5 minutes), 312 nm (5 and 10 minutes), and 365 nm (30 minutes) using Spectronics Corporation (Westbury, NY) hand-held lamps.…”

Stoichiometry for drug potentiation of a pentameric ion channel

“…In accord, the side chains of the substituted residues in the PNU-resistant mutant extend into this cavity and increase the EC 50 for potentiation ∼30-fold (15). Anesthetics, alcohols, and neurosteroids are thought to bind to a similar location in GABA A and glycine receptors (21-23), photoactivatable hydrophobic probes label nearby residues in the Torpedo acetylcholine receptor (24)(25)(26), and X-ray structures of a prokaryotic homolog show anesthetics interacting with residues lining the cavity (27). This intrasubunit site in α7 is distinct from the site for the potentiator ivermectin, which spans transmembrane α-helices from adjacent subunits in the invertebrate Cys-loop receptor, GluCl (28).…”

Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [3H]Allyl m-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate

“…Photoaffinity labeling studies have identified three classes of binding sites for allosteric modulators in the Torpedo nAChR TMD: 1) sites in the ion channel for "classical" cationic channel blockers, including chlorpromazine (Revah et al, 1990;Chiara et al, 2009) and tetracaine (Gallagher and Cohen, 1999), as well as uncharged, hydrophobic drugs, including the general anesthetics etomidate and propofol (Pratt et al, 2000;Ziebell et al, 2004;Nirthanan et al, 2008;Hamouda et al, 2011;Jayakar et al, 2013); 2) a site at the g2a subunit interface that binds positive (Nirthanan et al, 2008) and negative modulators (Hamouda et al, 2011;Jayakar et al, 2013); and 3) a site for negative modulators, including halothane and propofol, within the d subunit helix bundle (Chiara et al, 2003;Arevalo et al, 2005;Hamouda et al, 2008;Jayakar et al, 2013).…”