Concatemers to re-investigate the role of α5 in α4β2 nicotinic receptors

Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, β2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4β2 and α4β2α5 receptors and characterized their properties by two-electrodes voltage-clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and β2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.

Keywords: Biochemical pharmacology; Concatemers; Ligand-gated ion channels; Neuropharmacology; Nicotinic receptors.

Fig. 1

A robust concatemer design. a Schematic of the concatemers’ arrangement. Each subunit is depicted by an oval, with the first subunit in the N-terminal having its signal peptide as a circle. Linkers are depicted with a black line. b Anti-GFP Western blotting of whole extracts of HEK cells expressing the designated concatemer. c Sample traces recorded on oocytes expressing the 3(α4) (blue), 3(β2) (red), α5_first (dark green) and α5_last (light green) concatemers. For the 3(α4), ACh concentration was 100 µM, and for the 3(β2) and the α5, ACh concentration was 10 µM. d ACh concentration–response curves of the currents recorded for the four concatemers. Points are mean ± SD, with n ≥ 7

Fig. 2

Rotation direction of the 3(α4) concatemer. a Potential integrity of the NS9283 binding site depending on the position of the VFL triple-mutation. In the WT, NS9283 binds at the α4–α4 interface. In the triple-mutation VFL situation on all three α4 subunits, the NS9283 binding site is impaired and no potentiation is detected (Ahring 2017). When the VFL mutation is introduced in the middle α4 subunit (“SU3”) the NS9283 binding site is predicted to be intact. When it is introduced in the first (“SU1”) or the last (“SU5”) α4 subunit, pentamers that adopt an orientation that makes the VFL residues pointing in the NS9283 binding site should not be potentiated anymore. b ACh concentration–response curves of WT, SU1, SU3, SU5 and SU1 + SU5 constructs show that all mutants display gain-of-function phenotypes towards ACh, as previously described (Ahring 2017). Points are mean ± SD, with n ≥ 3. Unlike the original study, we choose a test ACh concentration that would allow to observe clear potentiation for the 3 constructs: 0.1 µM which is around the EC₁₀ of SU3, SU5 and SU1 + SU5, and EC₀₅ of SU1. c Example traces of NS9283 potentiation of the WT and the VFL mutants. Vertical bar is 100 nA, horizontal bar is 10 s. d Concentration–response curves of the potentiation of 0.1 µM ACh currents by NS9283. A dash line is drawn to figure 100%. Values measured for 100 µM NS9283 were not included in the curve fit because they were reproducibly lower than the 30 µM values, possibly due to a channel block effect, but they are still displayed as empty circles. Points are mean ± SD with n ≥ 3. * for SU1 + SU5, a Student’s t test found those two values to be non-statistically different

Fig. 3

The orthosteric site. a The nicotine binding pocket in the α4β2 X-ray structure visualized using PyMol (Morales-Perez 2018, pdb 5KXI). α4 subunit is shown in blue cartoon representation, β2 in red cartoon representation. Loop B and Loop C are labeled, Trp 156 is shown in orange stick representation and nicotine is depicted in yellow stick representation. b ACh concentration–response curves for the α5 concatemers WT and W169A mutant. Points are mean ± SD with n ≥ 3. c Sample traces of the effect of saturating concentrations of ACh and Sazetidine-A (Saz-A) on a single oocyte for the three concatemers. d Concentration–response curves for Saz-A of the three concatemers. Currents were normalized with the maximal current recorded with ACh for each cell. Points are mean ± SD with n ≥ 6

Fig. 4

Galantamine and progesterone fail to selectively modulate α5-containing concatemers. a Sample traces of Galantamine effect on the 3(α4) concatemer. Three protocols are displayed for the same cell: a 3-s pre-application, a 20-s pre-application and a co-application of galantamine with a sub-saturating ACh concentrations, showing no effect on the peak current. b Normalized peak currents obtained with the three protocols for the three concatemers. The apparent potentiation of 3(α4) is not significant as compared with the 3(β2). Displayed are mean ± SD with n ≥ 3. “n.s.” indicates that a one-way ANOVA with a Tukey’s post hoc test was performed and found the values not statistically different from each other. c Sample traces of progesterone inhibition on the three concatemers. For each cell, a control application of 17 mM of Ethanol was perform to account for the ethanol concentration used for progesterone solubilization. d Percentage inhibition of 30 µM ACh-evoked currents by 3.14 mM progesterone. Data on the right are corrected by the ethanol effect for each cell at 17 mM. The corrected values for progesterone inhibition at 3.14 µM are similar for the three concatemers with n ≥ 3: 63.0 ± 12% for 3(α4), 71.7 ± 12% for 3(β2) and 61.7 ± 12% for α5_first. “n.s.” indicates that a one-way ANOVA with a Tukey’s post hoc test was performed and found the values not statistically different from each other

Fig. 5

Apparent lack of functional effects of the α5-D398N mutation. a Anti-GFP western blot of whole cell extracts of HEK cells expressing the WT and D398N α5 concatemers. b Scattered dot-plot of maximal currents recorded in oocytes expressing the WT and D398N mutant α5 concatemers. Measurements were performed on injections series where the WT and mutant constructs were injected on the same oocytes batches and recorded the same day. Each dot corresponds to a single cell with n ≥ 10. “n.s.” indicates that a Student’s t test was performed and found the values not statistically different from each other. c ACh concentration–response curves for the α5 concatemers WT and D398N mutant. Points are mean ± SD with n ≥ 4. d Sample traces of responses to sub-saturating and saturating ACh concentrations for the WT (green) and D398N (purple) α5 concatemers. Traces were normalized to the peak to facilitate comparison. Oocytes from the same animal were compared. e Mean residual current observed after 5 s application of saturating ACh (10 µM) for the WT and D398N α5 concatemers. Oocytes from the same animal were compared with n ≥ 3. Displayed are mean ± SD. “n.s.” indicates that a Student’s t test was performed and found the values not statistically different from each other

Fig. 6

Irreversible channel-block of α5-nAChR by MTSEA. a, b Sample trace of the effect of MTSEA (200 µM) on the 3(α4) and α5 concatemers during a 20 s co-application with ACh and after 5 min wash. c Sequence alignment of the M2 transmembrane segment for α4, β2 and α5. Arrows indicate the cysteine residue in 2′ mutated in α5. d View from the top of the pore of the α4β2 nAChR X-ray structure visualized using PyMol (Morales-Perez 2018 pdb 5KXI). The α-carbon of each of the 2′ residues is depicted as yellow spheres. e Mean irreversible inhibition after a 20 s MTSEA (200 µM) + ACh(3 µM) application for the 3(α4), 3(β2), α5 and α5-C261S concatemers. Displayed are mean ± SD with n ≥ 4. *** Statistically different from α5_first (one-way ANOVA with Dunnett’s test using α5 as control group, p < 0.001)

Fig. 7

Evaluation of α5 content in pentamers from loose subunits. a, b Sample traces of the effect of MTSEA on the 3(α4) and α5 concatemers during a 30 s 1 mM co-application with ACh and after 5 min wash. c Scattered dot-plot presenting the permanent inhibition measured for each cell injected with the indicated DNA ratio and with n ≥ 4. One-way ANOVA with Dunnett’s test using 1:1:0 as control group found those values not different from the 1:1:0 ration (n.s.) or statistically different with p < 0.001 (***)