Additional Acetylcholine (ACh) Binding Site at α4/α4 Interface of (α4β2)2α4 Nicotinic Receptor Influences Agonist Sensitivity

Abstract

Nicotinic acetylcholine receptor (nAChR) ␣4 and ␤2 subunits assemble in two alternate stoichiometries to produce (␣4␤2) 2 ␣4 and (␣4␤2) 2 ␤2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at ␣4(؉)/␤2(؊) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either ␣4 or ␤2 as auxiliary subunits produces isoform-specific ␣4(؉)/␣4(؊) or ␤2(؉)/␤2(؊) interfaces. Using fully concatenated (␣4␤2) 2 ␣4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the ␣4(؉)/ ␣4(؊) interface that accounts for isoform-specific agonist sensitivity of the (␣4␤2) 2 ␣4 nAChR. The additional site resides in a region that also contains a potentiating Zn 2؉ site but is engaged by agonists to contribute to receptor activation. By engineering ␣4 subunits to provide a free cysteine in loop C at the ␣4(؉)␣4(؊) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the ␣4(؉)/(␣4(؊) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the ␣4(؉)/ ␣4(؊) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (␣4␤2) 2 ␣4 nAChRs.

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

“…The biphasic effect arises from the high and low sensitivity component. The high sensitivity component is appointed to the unaltered α2(+)/β2(−) interfaces, whereas the low sensitivity component is due to the impaired α2(+)/α2(−) interface (29). To the contrary, this mutation had no effect on either the EC 50 or the desensitization kinetics of the α2 W84A β2 HS subtype compared with the WT HS receptor (Tables S2 and S3 and Fig.…”

“…For that reason, we constructed the α2 mutant W84A and coexpressed it in Xenopus oocytes with the β2 WT nAChR subunit in 10:1 or in 1:10 RNA ratios, thus expressing solely the LS or the HS subtype, respectively (27). Trp84 is a highly conserved residue on the complementary side of the ligand binding site that in the α2-Epi structure was found to interact with the ligand directly and its role in the binding site of other nAChRs has been evaluated in numerous studies (29,39,40) (Fig. 4 A and B).…”

“…In the case of α4β2 nAChRs, the LS and HS subtypes display differential ligand specificity, unitary conductance and desensitization kinetics (28). It has been shown that these differences originate from the altered stoichiometry, since the LS subtype has, in addition to the α4(+)/β2(-) ligand binding sites, another one at the α4(+)/α4(−) interface (29). To emphasize the possible clinical significance of the two subtypes, it has been shown that the stoichiometry of the α4β2 nAChR can be altered by chronic nicotine exposure (28,30,31) or by mutations (32).…”

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…4 G and H, Right). Notably, previous studies show that expressing the heteromeric α4β2 nAChR under conditions that favor an (α4β2) 2 α4 stoichiometry (three α4 and two β2 subunits) results in a receptor having two α4(+)/β2(−) interfaces with high agonist sensitivity and a third binding site at the α4(+)/α4(−) interface that displays low agonist sensitivity (59)(60)(61).…”

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

“…This is reflected by the biphasic ACh concentration-response curves in in vitro assays at cells expressing a heterogeneous population of a4b2 nAChRs [13][14][15][16]. The lower ACh sensitivity of the LS receptor is believed to arise from a recently discovered low-sensitivity ACh binding site located in the a4/a4 interface only present in this stoichiometry [17,18]. Intriguingly, the high-and low-sensitive ACh binding sites in the LS receptor display differential responses to various nAChR ligands [19][20][21].…”