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

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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.

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Abbreviations

ACh:

Acetylcholine

GABA:

γ-Amino butyric acid

GFP:

Green fluorescent protein

MTS:

Methanethiosulfonate

nAChR:

Nicotinic acetylcholine receptor

SCAM:

Substituted cysteine accessibility method

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Acknowledgements

This work was founded by the ERC Grant (No. 788974, Dynacotine), the ANR Grant (No. 17-CE11-0030 Nicofive), the “équipe FRM” (Fondation pour la Recherche Médicale) grant DEQ20140329497, a fellowship from the Fondation ARC to MSP and a European Commission Research Executive Agency individual fellowship to MG (Marie Sklodowska-Curie Action, Individual Fellowship 659371). The authors would like to thank Uwe Maskos, Stéphanie Pons, Morgane Besson, Akos Nemecz and Alexandre Mourot for critical reading of the manuscript.

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MSP, MG and P-JC designed the study. MSP, HB, NB and MG performed and analyzed the experiments. MSP, MG and P-JC wrote the manuscript with inputs of all authors.

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Correspondence to Marc Gielen or Pierre-Jean Corringer.

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Prevost, M.S., Bouchenaki, H., Barilone, N. et al. Concatemers to re-investigate the role of α5 in α4β2 nicotinic receptors. Cell. Mol. Life Sci. 78, 1051–1064 (2021). https://doi.org/10.1007/s00018-020-03558-z

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