Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor: ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED

Research output: Contribution to journalJournal articleResearchpeer-review

Standard

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; Quek, Gracia; Lewis, Trevor M; Qudah, Taima; von Arenstorff, Ida; Ambrus, Joseph I; Harpsøe, Kasper; Karim, Nasiara; Balle, Thomas; McLeod, Malcolm D; Chebib, Mary.

In: The Journal of Biological Chemistry, Vol. 288, No. 37, 13.09.2013, p. 26521-32.

Research output: Contribution to journalJournal articleResearchpeer-review

Harvard

Absalom, NL, Quek, G, Lewis, TM, Qudah, T, von Arenstorff, I, Ambrus, JI, Harpsøe, K, Karim, N, Balle, T, McLeod, MD & Chebib, M 2013, 'Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor: ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED', The Journal of Biological Chemistry, vol. 288, no. 37, pp. 26521-32. https://doi.org/10.1074/jbc.M113.475053

APA

Absalom, N. L., Quek, G., Lewis, T. M., Qudah, T., von Arenstorff, I., Ambrus, J. I., ... Chebib, M. (2013). Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor: ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED. The Journal of Biological Chemistry, 288(37), 26521-32. https://doi.org/10.1074/jbc.M113.475053

Vancouver

Absalom NL, Quek G, Lewis TM, Qudah T, von Arenstorff I, Ambrus JI et al. Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor: ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED. The Journal of Biological Chemistry. 2013 Sep 13;288(37):26521-32. https://doi.org/10.1074/jbc.M113.475053

Author

Absalom, Nathan L ; Quek, Gracia ; Lewis, Trevor M ; Qudah, Taima ; von Arenstorff, Ida ; Ambrus, Joseph I ; Harpsøe, Kasper ; Karim, Nasiara ; Balle, Thomas ; McLeod, Malcolm D ; Chebib, Mary. / Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor : ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED. In: The Journal of Biological Chemistry. 2013 ; Vol. 288, No. 37. pp. 26521-32.

Bibtex

@article{3d38c00551c842198acd131048522d66,
title = "Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor: ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED",
abstract = "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 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 α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(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 (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs.",
author = "Absalom, {Nathan L} and Gracia Quek and Lewis, {Trevor M} and Taima Qudah and {von Arenstorff}, Ida and Ambrus, {Joseph I} and Kasper Harps{\o}e and Nasiara Karim and Thomas Balle and McLeod, {Malcolm D} and Mary Chebib",
year = "2013",
month = "9",
day = "13",
doi = "10.1074/jbc.M113.475053",
language = "English",
volume = "288",
pages = "26521--32",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "37",

}

RIS

TY - JOUR

T1 - Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

T2 - ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED

AU - Absalom, Nathan L

AU - Quek, Gracia

AU - Lewis, Trevor M

AU - Qudah, Taima

AU - von Arenstorff, Ida

AU - Ambrus, Joseph I

AU - Harpsøe, Kasper

AU - Karim, Nasiara

AU - Balle, Thomas

AU - McLeod, Malcolm D

AU - Chebib, Mary

PY - 2013/9/13

Y1 - 2013/9/13

N2 - 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 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 α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(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 (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs.

AB - 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 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 α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(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 (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs.

U2 - 10.1074/jbc.M113.475053

DO - 10.1074/jbc.M113.475053

M3 - Journal article

C2 - 23893416

VL - 288

SP - 26521

EP - 26532

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 37

ER -

ID: 58012051