Dataset Information

Differential ?4(+)/(-)?2 Agonist-binding Site Contributions to ?4?2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

ABSTRACT: Two ?4?2 nicotinic acetylcholine receptor (?4?2-nAChR) isoforms exist with (?4)2(?2)3 and (?4)3(?2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of ?4(+)/(-)?2 agonist-binding sites. The LS isoform also contains a unique ?4(+)/(-)?4 site with lower agonist affinity than the ?4(+)/(-)?2 sites. However, the relative roles of the conserved ?4(+)/(-)?2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform ?4?2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of ?2 subunit (-)-face E-loop residues to their non-conserved ?4 subunit counterparts or vice versa (?2HQT and ?4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the ?4(+)/(-)?2 sites contribute equally to function, making identical ?2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, ?4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent ?4(+)/(-)?2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

SUBMITTER: Lucero LM

PROVIDER: S-EPMC4732226 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Differential α4(+)/(-)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

Lucero Linda M LM Weltzin Maegan M MM Eaton J Brek JB Cooper John F JF Lindstrom Jon M JM Lukas Ronald J RJ Whiteaker Paul P

The Journal of biological chemistry 20151207 5

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied ...[more]

PMID: 26644472

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Project description:Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Dataset Information

Differential ?4(+)/(-)?2 Agonist-binding Site Contributions to ?4?2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

Publications

Differential α4(+)/(-)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

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