Non-Agonist-Binding Subunit Interfaces Confer Distinct Functional Signatures to the Alternate Stoichiometries of the  4 2 Nicotinic Receptor: An  4- 4 Interface Is Required for Zn2+ Potentiation

Abstract

The ␣4␤2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The ␣4 and ␤2 nAChR subunits assemble into two alternate stoichiometries, (␣4) 2 (␤2) 3 and (␣4) 3 (␤2) 2 , which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn 2ϩ . We show that Zn 2ϩ exerts an inhibitory modulatory effect on (␣4) 2 (␤2) 3 receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (␣4) 3 (␤2) 2 receptors. Furthermore, Zn 2ϩ inhibition on (␣4) 2 (␤2) 3 nAChRs is voltage-dependent, whereas it is not on (␣4) 3 (␤2) 2 receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn 2ϩ . Zn 2ϩ inhibition is mediated by a site located on the ␤2(ϩ)/␣4(Ϫ) subunit interfaces on both receptor stoichiometries. ␣4 H195 and ␤2 D218 are key determinants of this site. Zn 2ϩ potentiation on (␣4) 3 (␤2) 2 nAChRs is exerted by a site that resides on the ␣4(ϩ)/␣4(Ϫ) of this receptor stoichiometry. ␣4 H195 on the (Ϫ) side of the ACh-binding ␣4 subunit and ␣4 E224 on the (ϩ) side of the non-ACh-binding ␣4 subunit critically contribute to this site. We also identified residues within the ␤2 subunit that confer voltage dependency to Zn 2ϩ inhibition on (␣4) 2 (␤2) 3 , but not on (␣4) 3 (␤2) 2 nAChRs.

Targeting α4β2 Nicotinic Acetylcholine Receptors in Central Nervous System Disorders: Perspectives on Positive Allosteric Modulation as a Therapeutic Approach

“…In contrast, other PAMs have been reported to significantly increase the efficacy of the agonist at the receptors, including dFBr [73], NS206 [82] and HEPES at HS receptors [79], with dFBr also exhibiting a minor effect on ACh potency [72,]. PAMs with marked effects on both potency and efficacy are LY-2087101 [80], the NS9283 analogue 5k [84] and Zn 2+ on LS receptors [91]. Also, as often observed with nAChR agonists (e.g.…”

“…[67]), several of the PAMs display a bell-shaped concentration-response relationship at the receptor. This includes HEPES at HS receptors [79], dFBr [74], galantamine [76], Zn 2+ [91] and atropine, scopolamine and physostigmine [70].…”

“…This point is acknowledged by the authors of this review; however, for readability in this review, NS9283 will be termed a PAM. Ubiquitous ion in the central nervous system [90,91] identified a4b2 PAMs, it is currently not clear how modulation of desensitization versus deactivation will translate into behavioural effects, but it is likely that differential modulation of receptor kinetics will impact central synaptic transmission and thereby their in vivo efficacy. This is further discussed below.…”

“…So far, binding sites/regions have been identified for NS9283 and NS206 [82], Zn 2+ [91], galantamine [93], 17b-estradiol [68], LY-2087101 [94] and HEPES [79]. As a result of its binding site, a given PAM may display selective action at one stoichiometry or differential effects at the two stoichiometries.…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Site-directed mutagenesis, in combination with heterologous expression, has however been used successfully to examine phenomena such as, subunit glycosylation [225][226][227], the role of disulfide-linked cysteines [80,228], cell surface receptor trafficking [214,229,230], interactions with agonists and antagonists [231][232][233], modulation by zinc [234,235] and by other allosteric modulators [199,200], calcium permeability [236] and channel gating [202,[237][238][239][240][241]. Analysis of mutated nAChRs has also provided insights into how subunit domains may move during receptor activation [241,242].…”