Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists

Abstract

Background and purpose: Despite growing evidence that inhibition of α6β2-containing (α6β2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6β2*-nAChRs has delayed the discovery of α6β2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6β2 properties, to enable pharmacological characterization and the identification of novel α6β2-selective antagonists.

Experimental approach: Different combinations of the α6, β2, β3, chimeric α6/3 and mutant β3(V273S) subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized α6β2*-nAChRs was determined with ¹²⁵I-epibatidine binding.

Key results: Functional channels were detected after co-transfection of α6/3, β2 and β3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed α6/3β2β3(V273S)-nAChR pharmacology was unique, and clearly distinct from α4β2-, α3β4-, α7- and α1β1δε-nAChRs. Antagonist potencies in inhibiting α6/3β2β3(V273S) -nAChRs was similar to their binding affinity for rat native α6β2*-nAChRs. Agonist affinities for α6β2*-nAChRs was higher than their potency in activating α6/3β2β3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3β2β3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel α6β2-selective antagonists.

Conclusions and implications: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native α6β2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, α6β2-selective antagonists. We also propose a pharmacophore model of α6β2 antagonists, which offers a starting point for the development of new smoking cessation agents.

Figure 4

Chemical structure of the novel α6β2*-nAChRs antagonists identified with the screening process (compounds 1–6).

Figure 1

Functional responses obtained with different combinations of wild type (α6, β2 and β3) and modified (α6/3, β3^V273S) nAChR subunits following transient transfection into HEK293T cells, as tested in the FLIPR Ca²⁺ influx assay. Upon addition of 500 µM acetylcholine (A) or 200 nM epibatidine (B) at 10 s, normalized responses (as % of basal fluorescence units) were recorded. Subunit combination labels are above the corresponding curves. Traces from the subunit combinations that were inactive or poorly active, including α6β2β3, α6β2β3^V273S, β2β3 and control plasmid transfection, overlie and are un-labelled. CoTxMII, cells were co-incubated with 10 µM α-conotoxin MII.

Figure 2

The figure shows the activation, desensitization and recovery from desensitization of human α6/3β2β^V273S-nAChRs stably expressed in HEK293 cells, as tested in the FLIPR Ca²⁺ influx assay. Cells were loaded with the fluorescent Ca²⁺ indicator dye FLUO-4-AM and transferred to the FLIPR platform for the measurement of increases in intracellular Ca²⁺. Increases in the relative fluorescence units represent increases in intracellular Ca²⁺. Following the first addition of 200 nM nicotine, there was a peak of fluorescence change, reflecting channel activation and slow desensitization. Addition of the same quantity of nicotine to the same cells (second addition) did not yield an effect, confirming receptor desensitization. When cells plate was extensively washed with the assay buffer, residual fluorescence disappeared and addition of 200 nM nicotine (third addition) caused a further fluorescence increase, revealing recovery from desensitization. Data are from a representative experiment repeated at least three times with similar results.

Figure 3

Functional calcium concentration response curves of some nicotinic ligands in a FLIPR Ca²⁺ influx assay, two-step addition protocol. (A) Representative concentration response curves to the agonists nicotine, varenicline, sazetidine A, cytisine and A-85380, in the first addition. The antagonists α-conotoxin MII, α-conotoxin MII[H9A;L15A], α-conotoxin PIA, methyllycaconitine, and DHβE did not significantly increase calcium response (data not shown). Data were expressed as percentage of the maximum response to nicotine. (B) Representative concentration response curves to the agonists nicotine, varenicline, sazetidine A, cytisine and A-85380 for the inhibition of channel activation induced by the subsequent addition of 200 nM nicotine. Data were expressed as percentage of the response in the absence of inhibitor. (C) Representative concentration response curves to the antagonists α-conotoxin MII, α-conotoxin MII[H9A;L15A], α-conotoxin PIA, methyllycaconitine, and dihydro-β-erythroidine for the inhibition of channel activation induced by the subsequent addition of 200 nM nicotine. Data were expressed as percentage of the response in the absence of inhibitor.

Figure 5

Compounds 1–3 aligned to the top scoring pharmacophore model generated with Phase. The model consists of three features: a positive ionizable (blue), a hydrogen bond-acceptor (pink) and an aromatic ring (orange).