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

Jingyi Wang,Aarati Sriram,Alexander Kuryatov,Jon Lindstrom,Paul J. Kenny,Zhuang Jin,Theodore M. Kamenecka

doi:10.1074/jbc.m115.646786

Abstract

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.

Highlights

In (␣4␤2)2␣4 nicotinic acetylcholine receptors, there is an agonist binding site at the ␣4/␣4 subunit interface
To avoid misleading nomenclature and provide nomenclature that reflects the mechanism of action, we suggest designating sazetidine and NS9283 as ACh binding site site-selective agonists (SSAgs)
NS9283 Potentiates ␣2* and ␣4* but Not ␣3* nAChRs—As reported by others, NS9283 augments activation of nAChRs containing three ␣2 or three ␣4 subunits per nAChR expressed in oocytes or human embryonic kidney tsA201 (HEK) cells but not ␣3* nAChRs [10, 12]

Summary

Background

In (␣4␤2)2␣4 nicotinic acetylcholine receptors, there is an agonist binding site at the ␣4/␣4 subunit interface. Neuronal nicotinic acetylcholine receptors containing ␣4, ␤2, and sometimes other subunits (␣4␤2* 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 ␣4 and ␤2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is ␣4 but not if it is ␤2. We show that ␣2, ␣3, ␣4, and ␣6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with ␣4 subunits, but ␤2 and ␤4 accessory subunits cannot. NS9283 is neither allosteric nor a modulator It is not allosteric because NS9283 acts as a selective agonist at the ACh binding site formed at the ␣4/␣4 interface [16, 17]. Functional impairment by blocking accessory sites suggests that accessory agonist sites exist and that they promote channel activation from the interface between several ␣ subunits and ␣4

Experimental Procedures

Results

Discussion