ABSTRACT␣4 and ␤2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the ␣4 to ␤2 ratio. In this study, injection of oocytes with 1:10 ␣4/␤2 subunit cDNA ratios favored expression of HS ␣4␤2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS ␣4␤2 receptors. The stoichiometry was inferred from the shifts in the ACh EC 50 values caused by Leu to Thr mutations at position 9Ј of the second transmembrane domain of ␣4 and ␤2. The 1:10 injection ratio produced the (␣4) 2 (␤2) 3 stoichiometry, whereas 10:1 injections produced the (

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.

Background and purpose: a4 and b2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (a4)2(b2)3 and (a4)3(b2)2 receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (a4)2(b2)3 and (a4)3(b2)2 receptors. Experimental approach: a4 and b2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (a4)2(b2)3 or (a4)3(b2)2 receptors. Using two-electrode voltage-clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca 2+ permeability, sensitivity to modulation by Zn 2+ and sensitivity to up-regulation by chaperone protein 14-3-3. Key results: We found that pentameric concatamers with a subunit arrangement of b2_a4_b2_a4_b2 or b2_a4_b2_a4_a4 were stable and functional in Xenopus oocytes. By comparison, when a4 and b2 were concatenated with a subunit order of b2_b2_a4_b2_a4 or b2_a4_a4_b2_a4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both b2_a4_b2_a4_b2 and b2_a4_b2_a4_a4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn 2+ modulation, Ca 2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (a4)2(b2)3 and (a4)3(b2)2 receptors respectively. Using these concatamers, we found that most a4b2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-berythroidine, demonstrate stoichiometry-specific potencies and efficacies. Conclusions and implications:We concluded that the a4b2 nicotinic ACh receptors produced with b2_a4_b2_a4_b2 or b2_a4_b2_a4_a4 pentameric constructs are valid models of non-linked (a4)2(b2)3 and (a4)3(b2)2 receptors respectively.

Sazetidine-A has been recently proposed to be a "silent desensitizer" of ␣4␤2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes ␣4␤2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of ␣4␤2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by ␣4␤2* and ␣6␤2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant ␣4␤2 nAChRs in more detail. We expressed the two alternative stoichiometries of ␣4␤2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both ␣4(2)␤2(3)and ␣4(3)␤2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of ␣4␤2 nAChR: it was a full agonist on ␣4(2)␤2(3) nAChRs, whereas it had an efficacy of only 6% on ␣4(3)␤2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant ␣4␤2 nAChRs but shows differential efficacy on ␣4␤2 nAChRs subtypes.

The ␣4␤2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The ␣4 and ␤2 nAChR subunits assemble into two alternate stoichiometries, (␣4) 2 (␤2) 3 and (␣4) 3 (␤2) 2 , which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn 2ϩ . We show that Zn 2ϩ exerts an inhibitory modulatory effect on (␣4) 2 (␤2) 3 receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (␣4) 3 (␤2) 2 receptors. Furthermore, Zn 2ϩ inhibition on (␣4) 2 (␤2) 3 nAChRs is voltage-dependent, whereas it is not on (␣4) 3 (␤2) 2 receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn 2ϩ . Zn 2ϩ inhibition is mediated by a site located on the ␤2(ϩ)/␣4(Ϫ) subunit interfaces on both receptor stoichiometries. ␣4 H195 and ␤2 D218 are key determinants of this site. Zn 2ϩ potentiation on (␣4) 3 (␤2) 2 nAChRs is exerted by a site that resides on the ␣4(ϩ)/␣4(Ϫ) of this receptor stoichiometry. ␣4 H195 on the (Ϫ) side of the ACh-binding ␣4 subunit and ␣4 E224 on the (ϩ) side of the non-ACh-binding ␣4 subunit critically contribute to this site. We also identified residues within the ␤2 subunit that confer voltage dependency to Zn 2ϩ inhibition on (␣4) 2 (␤2) 3 , but not on (␣4) 3 (␤2) 2 nAChRs.