Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity.

ABSTRACT

Nicotinic acetylcholine receptor (nAChR) α4 and ß2 subunits assemble in two alternate stoichiometries to produce (α4ß2)(2)α4 and (α4ß2)(2)ß2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/ß2(-) 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 ß2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or ß2(+)/ß2(-) interfaces. Using fully concatenated (α4ß2)(2)α4 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 (α4ß2)(2)α4 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 α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) 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 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 (α4ß2)(2)α4 nAChRs.