An additional ACh binding site at the α4/α4 interface of the (α4β2)2α4 nicotinic receptor influences agonist sensitivity

Abstract Nicotinic ACh receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)2α4 and (α4β2)2β2, which display different agonist sensitivities. Functionally-relevant, agonist binding sites are thought to be located at α4(+)/β2(-) 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 α4 or β2 as accessory subunits produces isoform-specific α4(+)/α4(-) or β2(+)/β2(-) interfaces. Using fully concatenated (α4β2)2α4 nAChRs in conjunction with structural modeling, chimeric receptors and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4β2)2α4 nAChR. The additional site resides in a region that also contains a potentiating Zn2+ site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)/α4(-) interface, we demonstrated that the ACh responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent MTSET or MTSEA. The findings suggest that agonist occupation of the site at the α4(+)/α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4β2)2α4 nAChRs.