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

Abstract

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.

Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

“…The additional agonist site on the (␣4␤2) 2 ␣4 receptor receptors plays a dominant role in determining the signature properties of this receptor type, as compared with the (␣4␤2) 2 ␤2 receptor (29 -31). When the additional agonist site is ablated, (␣4␤2) 2 ␣4 receptor displays (␣4␤2) 2 ␤2-like pharmacology (30,31), including sensitivity to agonists and allosteric modulators (11,20,21). Significantly, introducing ␣4F312A in one of the two agonist sites of the (␣4␤2) 2 ␤2 receptor reduced dFBr efficacy by ϳ50% (Fig.…”

“…However, although L617F decreased dFBr EC 50 , F312Y and Y309F increased it, compared with wild type ( To further confirm the role of the ␣4 subunit on dFBr potentiation of ␣4␤2 nAChRs and further establish that M3 encodes binding residues for dFBr, we introduced ␣4F312A sequentially into concatenated (␣4␤2) 2 ␣4 and (␣4␤2) 2 ␤2 receptors. Concatenated receptors have fixed stoichiometry and subunit arrangement, and these constructs have been shown to replicate the functional properties of the receptors assembled from loose ␣4 and ␤2 subunits (21).…”

“…The ␣4␤2 nAChR can assemble in two functional stoichiometries: one with three copies of ␣4 and two copies of ␤2 [(␣4␤2) 2 ␣4] and the other with two copies of ␣4 and three copies of ␤2 [(␣4␤2) 2 ␤2] (20). The alternate receptors differ in pharmacological properties, including sensitivity to agonists and allosteric modulators (20,21).…”

“…To test the effects of dFBr in the alternate ␣4␤2 receptors, we expressed fully concatenated receptors. We have shown previously that concatenated ␣4␤2 receptors replicate the properties of their non-linkedcounterparts (21).Concatenatedreceptorswereconstructed as ␣4␤2 receptors as previously described (21). To introduce mutations into the subunits of the concatemers, the mutations were first introduced in free subunits cloned into a modified pCI plasmid (Promega), and following sequence verification by double-stranded DNA sequencing (SourceBioscience, Oxford, UK), the subunits were ligated to the desired position in the concatemer using unique enzyme restriction sites (21).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…A more ambitious recent approach aimed at constraining subunit stoichiometry has been the generation of five-subunit concatemers [263,264]. Studies such as these have helped to confirm that differences in pharmacological properties (such as high and low agonist sensitivity, as discussed above) can be a consequence of alternative subunit stoichiometries, for example (α4)2(β2)3 and (α4)3(β2)2.…”