Identification of Amino Acids of the Torpedo Nicotinic Acetylcholine Receptor Contributing to the Binding Site for the Noncompetitive Antagonist [³H]Tetracaine

Abstract

[(3)H]Tetracaine is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds with high affinity in the absence of cholinergic agonist (K(eq) = 0.5 microM) and weakly (K(eq) = 30 microM) in the presence of agonist (i.e., to nAChR in the desensitized state). In the absence of agonist, irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine results in specific photoincorporation of [(3)H]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [(3)H]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [(3)H]tetracaine were subjected to enzymatic digestion, and peptides containing (3)H were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [(3)H]tetracaine specifically labeled two sets of homologous hydrophobic residues (alphaLeu(251), betaLeu(257), gammaLeu(260), and deltaLeu(265); alphaVal(255) and deltaVal(269)) as well as alphaIle(247) and deltaAla(268) within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [(3)H]tetracaine-binding site is located within the lumen of the closed ion channel and provides a definition of the surface of the M2 helices facing the channel lumen.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

“…3 H]TDBzletomidate photolabeling of ␣ and ␦ M2-9 and/or -13 for nAChRs in the resting state was fully inhibited by tetracaine, consistent with the location of the binding site of tetracaine in the lumen of the ion channel as defined by photolabeling (43). The PCP binding site within the ion channel in the desensitized state has not been determined, but based upon photolabeling studies with [ 3 H]chlorpromazine (48), it is likely to bind at the level of M2-2, -6, and -9.…”

“…units (summarized in Table 1). Photolabeling of ␣M2-10 in the resting state was not inhibited by tetracaine, a resting state selective channel blocker that itself photolabels M2-9 and/or -13 in each subunit (43); and in the desensitized state, photolabeling of ␣M2-10 was not inhibited by PCP, which also binds in the ion channel although at an undetermined site. In contrast, tetracaine reduced photolabeling of ␣M2-13 and ␦M2-13 by Ͼ80%, and in the desensitized state PCP reduced labeling of ␣M2-9 and ␦M2-9 by Ͼ80%.…”

“…Residue ␣M2-10, labeling of which was not inhibited by tetracaine but was potentiated by PCP or proadifen, is colored red. Residues photolabeled by [ 3 H]tetracaine (43) are boxed. B and C, views of the T. californica nAChR homology model (␣, gold; ␤, blue; ␥, green; ␦, magenta) from perspectives parallel to the membrane surface (B) or looking down the channel from the extracellular side (C).…”

Enhanced Inhibition of a Mutant Neuronal Nicotinic Acetylcholine Receptor by Agonists: Protection of Function by (E)-N-Methyl-4-(3-pyridinyl)-3-butene-1-amine (TC-2403)

“…‫,ء‬ p Ͻ 0.05; ‫,ءء‬ p Ͻ 0.01; ‫,ءءء‬ p Ͻ 0.001. (Papke and Oswald, 1989;Takayama et al, 1989;Gallagher and Cohen, 1999;Middleton et al, 1999;Blanton et al, 2000). Therefore, these two agents should serve as effective probes for channel-associated sites and in fact may distinguish between sites associated with different .…”

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).…”