Noncompetitive Antagonist Binding Sites in the Torpedo Nicotinic Acetylcholine Receptor Ion Channel. Structure−Activity Relationship Studies Using Adamantane Derivatives

Abstract

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4β2 and α3β4 subtypes

“…To determine receptor selectivity, the effect of varenicline on [ 3 H] MLA (4.1 nM) binding to hα7 nAChRs, on [ 3 H]epibatidine (4.6 nM) binding to hα3β4 nAChRs, and on [ 3 H]cytisine (9.1 nM) binding to hα4β2, hα4β4, and Torpedo nAChRs, respectively, was studied as previously described [19][20][21][22]. To determine whether varenicline interacts with the Torpedo and hα4β2 nAChR ion channels, additional studies were conducted using [ 3 H]TCP (20 nM) [21] and [ 3 H]imipramine (13 nM) [23].…”

“…To determine whether varenicline interacts with the Torpedo and hα4β2 nAChR ion channels, additional studies were conducted using [ 3 H]TCP (20 nM) [21] and [ 3 H]imipramine (13 nM) [23]. The effect of varenicline on [ 3 H]cytisine, in the absence (nAChRs are in the resting but activatable state) and presence of 200 μM proadifen [24], and [ 3 H]TCP binding, in the presence of 1 mM CCh (nAChRs are mainly in the desensitized state), was also determined as previously described [21,23] After incubation (2 h), nAChR-bound radioligand was separated from free radioligand by a filtration assay [19][20][21][22][23]. The concentrationresponse data were curve-fitted by nonlinear least squares analysis using the Prism software (GraphPad Software, San Diego, CA).…”

Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates

“…However, there is much evidence that the ion pore admits large molecules only from the extracellular side and that depth of penetration into the pore is a function of molecular size [2,3,6,[29][30][31][32][33][34]. As a result, it seems clear that the pore must be shaped like a funnel that opens toward the extracellular side (Fig.…”

“…This structure has been suggested to be a progenitor of pentameric ion channels [30]. We previously used the five pore-lining alpha helices in this structure as a template for aligning the pore-lining transmembrane segment 2 (TM2) in models of GABAAR, GlyR, and nAChR ion channels [1,[6][7][8]18,32]. The MscL structure also contains five outer alpha helices that contribute to the ion channel only as the funnel-shaped pore widens outward on the extracellular side.…”

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

“…To characterize mTFD-MPAB interactions with the transmitter binding sites, we compared its effects on the equilibrium binding of [ H]TCP, a PCP analog; Katz et al, 1997;Arias et al, 2003Arias et al, , 2006. Binding assays were performed in the presence of Carb, an agonist that stabilizes nAChRs in the desensitized state, or a-BgTx, a competitive antagonist that stabilizes the nAChR in the resting state.…”