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Covalent trapping of methyllycaconitine at the ?4-?4 interface of the ?4?2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

ABSTRACT: The ?4?2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the ?4?2 nAChR, (?4)2(?2)3 and (?4)3(?2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and ?4?2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the ?4-?4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an ?4?2 receptor containing an ?4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (?4)3(?2)2 stoichiometry, indicating that MLA binds to the ?4-?4 interface of the (?4)3(?2)2 and providing direct evidence of ligand binding to the ?4-?4 interface. Consistent with other studies, we propose that the ?4-?4 interface is a structural target for potential therapeutics that modulate (?4)3(?2)2 nAChRs.

SUBMITTER: Absalom NL

PROVIDER: S-EPMC3772200 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4β2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

Absalom Nathan L NL Quek Gracia G Lewis Trevor M TM Qudah Taima T von Arenstorff Ida I Ambrus Joseph I JI Harpsøe Kasper K Karim Nasiara N Balle Thomas T McLeod Malcolm D MD Chebib Mary M

The Journal of biological chemistry 20130726 37

The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4β2 nAChRs in the Xenopus oocyte expression sy ...[more]

PMID: 23893416

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Project description:Nicotine, the principal reinforcing compound in tobacco, acts in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). This review summarizes our current knowledge regarding how the α5 accessory nAChR subunit, encoded by the CHRNA5 gene, differentially modulates α4β2* and α3β4* receptors at the cellular level. Genome-wide association studies have linked a gene cluster in chromosomal region 15q25 to increased susceptibility to nicotine addiction, lung cancer, chronic obstructive pulmonary disease, and peripheral arterial disease. Interestingly, this gene cluster contains a non-synonymous single-nucleotide polymorphism (SNP) in the human CHRNA5 gene, causing an aspartic acid (D) to asparagine (N) substitution at amino acid position 398 in the α5 nAChR subunit. Although other SNPs have been associated with tobacco smoking behavior, efforts have focused predominantly on the D398 and N398 variants in the α5 subunit. In recent years, significant progress has been made toward understanding the role that the α5 nAChR subunit-and the role of the D398 and N398 variants-plays on nAChR function at the cellular level. These insights stem primarily from a wide range of experimental models, including receptors expressed heterologously in Xenopus oocytes, various cell lines, and neurons derived from human induced pluripotent stem cells (iPSCs), as well as endogenous receptors in genetically engineered mice and-more recently-rats. Despite providing a wealth of available data, however, these studies have yielded conflicting results, and our understanding of the modulatory role that the α5 subunit plays remains incomplete. Here, we review these reports and the various techniques used for expression and analysis in order to examine how the α5 subunit modulates key functions in α4β2* and α3β4* receptors, including receptor trafficking, sensitivity, efficacy, and desensitization. In addition, we highlight the strikingly different role that the α5 subunit plays in Ca2+ signaling between α4β2* and α3β4* receptors, and we discuss whether the N398 α5 subunit variant can partially replace the D398 variant.

Dataset Information

Covalent trapping of methyllycaconitine at the ?4-?4 interface of the ?4?2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

Publications

Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4β2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

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