Potentiation of a neuronal nicotinic receptor via pseudo-agonist site

Mazzaferro, Simone; Bermúdez, Isabel; Sine, Steven M.

doi:10.1007/s00018-018-2993-7

Cited by 19 publications

(17 citation statements)

References 59 publications

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“…Combined data strongly support that NS9283 binds specifically at the α4-α4 interface [27]. Single channel measurements also showed that NS9283 potentiation was sensitive to mutations performed on the principal face of the β2 subunit [22].…”

Section: Rotational Direction Of Concatemerssupporting

confidence: 54%

“…Several pentameric concatemers of heteromeric receptors have been described incorporating the α4β2 and α3β4 subunits [11,[14][15][16][17][18][19][20][21][22], as well as the α3β4α5 subunits [11,19]. In contrast, a single study described the α4β2α5 combination, with an arrangement (β2α4α5α4α4/β2) where only one α4β2 pair (and therefore one canonical α4/β2 binding site) is present in the pentamer and that are endowed with weak ACh-gated currents (less than 50nA in oocytes [19]).…”

Section: Main Text Introductionmentioning

confidence: 99%

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

Prevost¹,

Bouchenaki²,

Barilone³

et al. 2020

Cell. Mol. Life Sci.

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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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Section: Rotational Direction Of Concatemerssupporting

confidence: 54%

Section: Main Text Introductionmentioning

confidence: 99%

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

Prevost¹,

Bouchenaki²,

Barilone³

et al. 2020

Cell. Mol. Life Sci.

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“…An excess of α4 over β2 cDNAs has been shown to increase expression of (α4) 3 (β2) 2 over the (α4) 2 (β2) 3 stoichiometry in BOSC 23 cells (Carignano et al, 2016). To ensure a pure population of the (α4) 3 (β2) 2 stoichiometry, the cDNAencoding 14-3-3η was added to the transfection mix (Mazzaferro et al, 2017(Mazzaferro et al, , 2019(Mazzaferro et al, , 2021. The amounts of transfected α4 and β2…”

Section: Methodsmentioning

confidence: 99%

Stoichiometry‐selective modulation of α4β2 nicotinic ACh receptors by divalent cations

Mazzaferro

Strikwerda

Sine

2022

British J Pharmacology

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Background and Purposeα4β2 nicotinic ACh receptors (nAChRs) comprise the most abundant class of nAChRs in the nervous system. They assemble in two stoichiometric forms, each exhibiting distinct functional and pharmacological signatures. However, whether one or both forms are modulated by calcium or magnesium has not been established.Experimental ApproachTo assess the functional consequences of calcium and magnesium, each stoichiometric form was expressed in clonal mammalian fibroblasts and single‐channel currents were recorded in the presence of a range of ACh concentrations.Key ResultsIn the absence of divalent cations, each stoichiometric form exhibits high unitary conductance and simple gating kinetics composed of solitary channel openings or short bursts of openings. However, in the presence of calcium and magnesium, the conductance and gating kinetics change in a stoichiometry‐dependent manner. Calcium and magnesium reduce the conductance of both stoichiometric forms, with each cation producing an equivalent reduction, but the reduction is greater for the (α4)2(β2)3 form. Moreover, divalent cations promote efficient channel opening of the (α4)3(β2)2 stoichiometry, while minimally affecting the (α4)2(β2)3 stoichiometry. For the (α4)3(β2)2 stoichiometry, at high but not low ACh concentrations, calcium in synergy with magnesium promote clustering of channel openings into episodes of many openings in quick succession.Conclusion and ImplicationsModulation of the α4β2 nAChR by divalent cations depends on the ACh concentration, the type of cation and the subunit stoichiometry. The functional consequences of modulation are expected to depend on the regional distributions of the stoichiometric forms and synaptic versus extrasynaptic locations of the receptors.

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“…Several whole genome duplications occurred at the beginning of vertebrate evolution, which probably were the origin of the initial diversity in many gene families [47][48] Meanwhile, residues of the vestibule β4-β5 loop interact with passing Ca 2+ in the mammalian α9α10 nAChRs [42], and the vestibule of α7 receptors exist a negative allosteric site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC [46], justifying the positive selection on the β4-β5 loop of the α3α6 tribe has important role in the formed receptors, such as Ca 2+ permeability or the allosteric modulation. At the β2-α4 interface in (α4) 2 (β2) 3 receptors, H137 of α4 is a NS9283-enhanced allosteric site [51], which is located at the beginning of loop-E (β5 strand). It is unexpected that some positively selected sites exist on the β2 strand (loop-D) of α3α6 tribe and the β5 strand (loop-E) of α2α4 tribe.…”

Section: Functional Differentiation In Neuronal Nachrs Subunit Tribesmentioning

confidence: 99%

Analysis of the Functional Differentiation Sites in Vertebrate Neuronal Nicotinic Acetylcholine Receptor Subunits

Zhao

Xin

et al. 2020

Preprint

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The nicotinic acetylcholine receptors (nAChR) belong to a large family of ligand-gated ion channels and are involved in the mediation of fast synaptic transmission. Each receptor is made up of five subunits that arrange symmetrically around a central pore. Despite the similarity in their sequences and structures, the properties of these subunits vary significantly. Thus, identifying the function-related sites specific to each subunit is essential for understanding the characteristics of the subunits and the receptors assembled by them. In this study, we examined the sequence features of the nine neuronal nAChRs subunits from twelve representative vertebrate species. Analysis revealed that all the subunits were subject to strong purifying selection in evolution, and each was under a unique pattern of selection pressures. At the same time, the functional constraints were not uniform within each subunit, with different domains in the molecule being subject to different selection pressures. Via evolutionary analyses, we also detected potential positive selection events in the subunits or subunit clusters, and identified the sites might be associated with the function specificity of each subunit. Furthermore, positive selection at some domains might contributed to the diversity of subunit function; for example, the β9 strand might be related to the agonist specificity of α subunit in heteromeric receptor and β4-β5 linker could be involved in Ca2+ permeability. Subunits α7, α4 and β2 subunits possess a strong adaptability in vertebrates. Our results highlighted the importance of tracking functional differentiation in protein sequence underlying functional properties of nAChRs. In summary, our work may provide clues on understanding the diversity and the function specificity of the nAChR subunits, as well as the receptors co-assembled by them.

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Potentiation of a neuronal nicotinic receptor via pseudo-agonist site

Cited by 19 publications

References 59 publications

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

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

Stoichiometry‐selective modulation of α4β2 nicotinic ACh receptors by divalent cations

Analysis of the Functional Differentiation Sites in Vertebrate Neuronal Nicotinic Acetylcholine Receptor Subunits

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