Pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 nicotinic acetylcholine receptors: subunit arrangement determines functional expression

alpha 4 and beta 2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors. alpha 4 and beta 2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (alpha 4)(2)(beta 2)(3) or (alpha 4)(3)(beta 2)(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, Ca2+ permeability, sensitivity to modulation by Zn2+ and sensitivity to up-regulation by chaperone protein 14-3-3. We found that pentameric concatamers with a subunit arrangement of beta 2_alpha 4_beta 2_alpha 4_beta 2 or beta 2_alpha 4_beta 2_alpha 4_alpha 4 were stable and functional in Xenopus oocytes. By comparison, when alpha 4 and beta 2 were concatenated with a subunit order of beta 2_beta 2_alpha 4_beta 2_alpha 4 or beta 2_alpha 4_alpha 4_beta 2_alpha 4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both beta 2_alpha 4_beta 2_alpha 4_beta 2 and beta 2_alpha 4_beta 2_alpha 4_alpha 4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn2+ modulation, Ca2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors respectively. Using these concatamers, we found that most alpha 4 beta 2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-beta-erythroidine, demonstrate stoichiometry-specific potencies and efficacies. We concluded that the alpha 4 beta 2 nicotinic ACh receptors produced with beta 2_alpha 4_beta 2_alpha 4_beta 2 or beta 2_alpha 4_beta 2_alpha 4_alpha 4 pentameric constructs are valid models of non-linked (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors respectively.