Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

Abstract

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for alpha-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [(3)H]-(+/-)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15nM and 1muM. These classes of sites exist in approximately equal numbers and all [(3)H]-(+/-)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.

Fig. 1

The equilibrium binding of [³H]-(±)-epibatidine to Torpedo membranes (0.5 µM in α-BTx binding sites) was measured by a centrifugation assay. (A) The dashed line represents the best fit for binding to a single population of sites with B_max = 0.88 µM and K_d = 34 nM. The solid line shows a better fit to a two component model with B_max1 = 0.56 µM, K_d1 = 13.5 nM, B_max2 = 0.38 µM and K_d2 = 0.29 µM. (B) A Scatchard transformation of the data in (A) with the solid line calculated using the parameters obtained from the two component model in (A).

Fig. 2

The binding of [³H]-(±)-epibatidine to Torpedo membranes (5 µM in α-BTx sites) was measured by equilibrium dialysis. [³H]-(±)-epibatidine binds to approximately twice as many sites as α-BTx and fitting by a two state model gave B_max1 = 4.96 µM, K_d1 = 10.4 nM, B_max2 = 5.95 µM, K_d2 = 0.98 µM. The curve for total binding (●) includes a non-specific binding (○) component of 0.0106 µM bound/µM free measured the presence of excess unlabelled ligand. Data are averaged from two independent experiments and are representative of five similar assays carried out using high concentrations of nAChR (2.5 – 5 µM in α-BTx sites).

Fig. 3

Displacement of radiolabelled agonist by unlabelled ligands. In each case the receptor concentration was 0.3 µM in α-BTx sites. (A) Displacement of [³H]ACh (1 µM) by ACh (○) was well described by a simple sigmoidal curve giving IC₅₀ = 0.60 µM and n_H = 1 whereas the effects of (±)epibatidine (■) was biphasic giving IC₅₀₍₁₎ = 0.148 µM, IC₅₀₍₂₎ = 8.05 µM and F_H = 0.528. Corresponding data for (+)-epibatidine (▲) gave IC₅₀₍₁₎ = 0.307 µM, IC₅₀₍₂₎ = 61.6 µM and F_H = 0.56. (B) Displacement of [³H]-(±)-epibatidine (0.5 µM) by ACh (○) was biphasic and curve fitting gave IC₅₀₍₁₎ = 0.36 µM, IC₅₀₍₂₎ = 11.5 µM and F_H = 0.644. In contrast displacement by (±)-epibatidine (■) can be described by a one-component fit with IC₅₀ = 0.47 µM and n_H = 1. (C) Displacement of [³H]-SbCh (1 µM) by ACh (○) was best described by a sigmoidal curve with IC₅₀ = 1.39 µM and n_H = 0.733. The effects of (±)-epibatidine (■) were described by a similar model with IC₅₀ = 2.34 µM and n_H = 0.598.

Fig. 4

The effects of receptor inhibitors on the binding of [³H]-(±)-epibatidine. [³H]-(±)-epibatidine (0.5 µM) was incubated with nAChR enriched membranes (0.3 µM in α-BTx sites) and the effects of d-tubocurarine (●), β-erythroidine (○), chlorpromazine HCl (▼) and MTPP-Br (◇) were measured using a centrifugation assay. Only d-tubocurarine caused significant displacement below 100 µM and the data were best fit by a sigmoidal competition curve with IC₅₀ = 22.1 µM and n_H = 0.681.