Mazzaferro_2011_J.Biol.Chem_286_31043

Nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits assemble in two alternate stoichiometries to produce (alpha4beta2)(2)alpha4 and (alpha4beta2)(2)beta2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at alpha4(+)/beta2(-) 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 alpha4 or beta2 as auxiliary subunits produces isoform-specific alpha4(+)/alpha4(-) or beta2(+)/beta2(-) interfaces. Using fully concatenated (alpha4beta2)(2)alpha4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the alpha4(+)/alpha4(-) interface that accounts for isoform-specific agonist sensitivity of the (alpha4beta2)(2)alpha4 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 alpha4 subunits to provide a free cysteine in loop C at the alpha4(+)alpha4(-) 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 alpha4(+)/(alpha4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the alpha4(+)/alpha4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (alpha4beta2)(2)alpha4 nAChRs.