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Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine ?4?2 receptors: unique role of halogen bonding revealed.

ABSTRACT: The ?4?2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of ?4?2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at ?4?2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for ?4?2 receptors. Crystal structures of five agonists with efficacies at ?4?2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

SUBMITTER: Rohde LA

PROVIDER: S-EPMC3281707 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4β2 receptors: unique role of halogen bonding revealed.

Rohde Line Aagot Hede LA Ahring Philip Kiær PK Jensen Marianne Lerbech ML Nielsen Elsebet Østergaard EØ Peters Dan D Helgstrand Charlotte C Krintel Christian C Harpsøe Kasper K Gajhede Michael M Kastrup Jette Sandholm JS Balle Thomas T

The Journal of biological chemistry 20111213 6

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A c ...[more]

PMID: 22170047

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Project description:Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (?4)2(?2)3) or low-sensitivity (LS) (?4)3(?2)2) isoforms of human ?4?2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-h?4?2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS ?4?2-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 ?4?2-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 ?4(+)/(-)?4 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 ?2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP ?4?2-nAChRs. Thus, recruitment of the ?4(+)/(-)?4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of ?4(+)/(-)?2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two ?4?2-nAChR isoforms, and demonstrate that HS versus LS ?4?2-nAChR activity can be selectively manipulated using pharmacological approaches. Since ?4?2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

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Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine ?4?2 receptors: unique role of halogen bonding revealed.

Publications

Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4β2 receptors: unique role of halogen bonding revealed.

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