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J Biol Chem. 2015 May 29;290(22):13907-18. doi: 10.1074/jbc.M115.646786. Epub 2015 Apr 13.

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors.

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From the Department of Neuroscience, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Department of Molecular Therapeutics at the Scripps Research Institute, Scripps, Florida 33458, and.
Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
From the Department of Neuroscience, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104,


Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.


MTSEA alkylation; NS9283; acetylcholine; drug development; electrophysiology; mutagenesis; nicotinic acetylcholine receptors (nAChR); pharmacology; receptor structure-function

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