Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Abstract

Two ␣4␤2 nicotinic acetylcholine receptor (␣4␤2-nAChR) isoforms exist with (␣4) 2 (␤2) 3 and (␣4) 3 (␤2) 2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of ␣4(؉)/(؊)␤2 agonist-binding sites. The LS isoform also contains a unique ␣4(؉)/ (؊)␣4 site with lower agonist affinity than the ␣4(؉)/(؊)␤2 sites. However, the relative roles of the conserved ␣4(؉)/(؊)␤2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform ␣4␤2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of ␤2 subunit (؊)-face E-loop residues to their non-conserved ␣4 subunit counterparts or vice versa (␤2HQT and ␣4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125 I-mAb 295 binding and was used to define function/nAChR. If the ␣4(؉)/(؊)␤2 sites contribute equally to function, making identical ␤2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, ␣4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent ␣4(؉)/(؊)␤2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

Isoform‐specific mechanisms of a3b4*‐nicotinic acetylcholine receptor modulation by the prototoxin lynx1

“…There was, however, one exception: at the highest lynx1 mRNA coinjection only, function per cell‐surface (α3β4) 2 β4nAChR increased significantly. Of interest, function per receptor levels in the absence of lynx1 are <5 μA/fmol across all α3β4*‐nAChR isoforms, which is much lower than that previously measured for α4β2*‐nAChR using a similar approach (48, 74). Given the greater bursting behavior and slower macroscopic desensitization of α3β4*‐ (references cited above, data from this study) vs .…”

“…This explanation also fits with a previous observation that β4‐nAChR subunits lack endoplasmic reticulum–retention/retrieval motifs that were found within β2‐nAChR subunits, and have an endoplasmic reticulum–export motif missing from β2 subunits (75), which results in increased surface expression—potentially at the expense of quality control. This explanation is further reinforced by the fact that surface expression of α3β4*‐nAChR—especially of the isoform that contains 3 β4 subunits—is much higher than that of α4β2*‐nAChR as measured in our previous studies (48, 74), and, as demonstrated in the current study, removal of these putative quiescent (α3β4) 2 β4‐nAChRs from the cell surface can be reversed by GPI anchor cleavage, which suggests a possible cell‐surface role for lynx1 in this quality control phenomenon.…”

“…For example, several studies, including our own, demonstrate that binding of both divalent cations (88, 92) and agonists at α(+)/(−)α interfaces can profoundly affect efficacy when measured at the macroscopic (48, 89, 93, 94) and single‐channel levels (92). Second, there is a demonstrated functional role for residues that are located on the conserved agonist‐binding E‐loop of α(−) subunits, both at agonist binding sites and outside of them (74, 89, 95, 96).…”

Molecular and cellular characterization of nicotinic acetylcholine receptor subtypes in the arcuate nucleus of the mouse hypothalamus

“…In both the brain and the periphery nicotine acts on nicotinic acetylcholine receptors (nAChR), cation channels made up of a combination α-and β-type subunits arranged as a pentamer around a central pore (Albuquerque, Pereira, Alkondon, & Rogers, 2009;Morales-Perez, Noviello, & Hibbs, 2016). The identity of the individual subunits and the stoichiometry of α and β subunits within an assembled nAChR determine many channel properties, including sensitivity to both endogenous and exogenous ligands, kinetics of ligand binding, the amplitude and identity of ionic currents that flow through the channel, and channel open time (George et al, 2017;Lucero et al, 2015;Mazzaferro, Bermudez, & Sine, 2016;Stokes & Papke, 2012). In addition, each nicotinic subunit is expressed in distinct, though overlapping, patterns in many cell types throughout the brain (Sargent, 1993).…”

The fifth subunit of the (α4β2)2β2 nicotinic ACh receptor modulates maximal ACh responses

“…The α4β2 nAChR comprises two α4β2 pairs and a fifth subunit that can be β2 or α4, and this subunit difference produces two alternate receptor isoforms, the (α4β2) 2 β2 and (α4β2) 2 α4 nAChRs (Nelson et al , ; Moroni et al , ) (Figure A). The alternative receptors display strikingly different sensitivities to activation by ACh and other agonists (Nelson et al , ; Moroni et al , ; Harpsøe et al , ; Mazzaferro et al , ; Timmermann et al , ; Absalom et al , ; Lucero et al , ), high‐affinity desensitization (Marks et al , ; Benallegue et al , ), sensitivity to allosteric modulators (Moroni et al , ; Olsen et al , ; Alcaino et al , ; Jin et al , ) and single‐channel properties (Mazzaferro et al , ). These differences are accounted for partly by an additional operational agonist site in the (α4β2) 2 α4 stoichiometry housed by the interface between the fifth subunit (an α4) and an adjacent α4 subunit (Harpsøe et al , ; Mazzaferro et al , ).…”