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.

The α5 Nicotinic Acetylcholine Receptor Subunit Differentially Modulates α4β2* and α3β4* Receptors

“…Lately, Prevost and colleagues used pentameric concatemer constructs for expression in Xenopus oocytes (Prevost et al, 2020). Similar to previous reports they observed no difference in ACh potency between human (α4β2) 2 β2 and (α4β2) 2 α5 receptors, whereas (α4β2) 2 α4 receptors showed a biphasic concentration response curve with an overall significantly reduced ACh potency.…”

Structural correlates of human muscle nicotinic acetylcholine receptor subunit assembly mediated by δ(+) interface residues

“…However, we cannot at this stage, completely rule out other alternatives. That would require the use of concatemers [54, Mazzaferro, 2014 #6892], a technique that has recently been used to show that loose subunits can lead to heterogenous receptor populations, although this was for α4β2α5 receptors [55]. Nevertheless, due to the robust currents recorded, no change in hill slope compared to WT and the requirement of a β subunit for cell surface expression [17,19], a reasonable and parsimonious assumption is that this receptor adopts a αδαδβ ordering.…”

Alterations in nicotinic receptor alpha5 subunit gene differentially impact early and later stages of cocaine addiction: a translational study in transgenic rats and patients

“…Additional work will also now be needed to further understand how the α5SNP, which has been proposed to confer a partial loss of function of the receptor although the underlying mechanisms are still unclear (Maskos, 2020;Prevost et al, 2020), may confer protection against CUD development by modifying the effects of cocaine at early stages of exposure, while a full absence of α5nAChRs rather seems to increase the risk to relapse after prior long-term exposure.…”

“…This 5SNP is very frequent in the general population and may induce a partial loss of function of the corresponding pentamer (Bierut et al, 2008;Frahm et al, 2011;Kuryatov et al, 2011) without altering the overall expression of the subunit, at least in rats (Forget et al, 2018). It does not modify the electrophysiological function of the receptor when expressed in concatemer in Xenopus oocytes (Prevost et al, 2020), and it lies away from agonists' binding site, suggesting that it affects other features of the receptor, but its precise mechanism of action is currently unknown (Maskos, 2020). Interestingly, in candidate gene association studies, the 5SNP has also been associated with a lower prevalence of CUD in humans (Grucza et al, 2008;Sherva et al, 2010).…”