Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement determines functional expression

Abstract

Background and purpose: alpha4 and beta2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors.

Experimental approach: alpha4 and beta2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (alpha4)(2)(beta2)(3) or (alpha4)(3)(beta2)(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.

Key results: We found that pentameric concatamers with a subunit arrangement of beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 were stable and functional in Xenopus oocytes. By comparison, when alpha4 and beta2 were concatenated with a subunit order of beta2_beta2_alpha4_beta2_alpha4 or beta2_alpha4_alpha4_beta2_alpha4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both beta2_alpha4_beta2_alpha4_beta2 and beta2_alpha4_beta2_alpha4_alpha4 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 (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively. Using these concatamers, we found that most alpha4beta2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-beta-erythroidine, demonstrate stoichiometry-specific potencies and efficacies.

Conclusions and implications: We concluded that the alpha4beta2 nicotinic ACh receptors produced with beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 pentameric constructs are valid models of non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively.

Figure 1

Diagram representing the (α4)₂(β2)₃ and (α4)₃(β2)₂ nAChR thought to be formed by the pentameric concatenated constructs shown underneath the diagrams. Dotted lines represent the synthetic linkers bridging the subunits.

Figure 2

Expression of pentameric concatamers with subunit order of β2_β2_α4_β2_α4 or β2_α4_α4_β2_α4 in Xenopus oocytes. (A) ACh evoked small inward currents in oocytes microinjected with 150 ng of β2_Q8β2_Q8α4_Q8β2_Q8α4 or β2_Q8α4_Q8α4_Q8β2_Q8α4 cRNA. For comparison the ACh EC₁₀₀ current responses of the corresponding non-linked receptors are included. Arrows indicate the start of the application of ACh. (B) Molecular mass of β2_Q8β2_Q8α4_Q8β2_Q8α4 or β2_Q8α4_Q8α4_Q8β2_Q8α4 proteins. Membrane homogenates prepared from oocytes injected with β2_Q8β2_Q8α4_Q8β2_Q8α4 or β2_Q8α4_Q8α4_Q8β2_Q8α4 cRNAs were resolved and then blotted and immunostained as described in Methods. Note the bands underneath the 310 and 290 kDa bands, which suggest cleavage of the pentameric constructs. (C) Western blot analysis of β2_AGSβ2_AGSα4_AGSβ2_AGSα4 and β2_AGSα4_AGSα4_AGSβ2_AGSα4 proteins indicated that the pentameric constructs were not cleaved. Total protein from oocytes injected with β2_AGSβ2_AGSα4_AGSβ2_AGSα4 and β2_AGSα4_AGSα4_AGSβ2_AGSα4 cRNAs was resolved by reducing SDS-PAGE on a NuPage 7% Tris Acetate gel. Immunoblot analysis was carried as described in Methods. The molecular mass of the concatenated constructs are shown next to the blots.

Figure 3

Expression of β2_α4_β2_α4_β2 and β2_α4_β2_α4_α4 in Xenopus oocytes. (A) Western blot analysis indicated that pentameric concatamers β2_α4_β2_α4_β2 and β2_α4_β2_α4_α4 are synthesized and stable when expressed heterologously in Xenopus oocytes. Total protein from oocytes injected with either β2_α4_β2_α4_β2 or β2_α4_β2_α4_α4 cRNAs was separated by reducing SDS-PAGE on a NuPage 7% Tris Acetate gel. (B) 1 mmol·L⁻¹ ACh (EC₁₀₀) evoked inward currents whose amplitude was concentration-dependent in oocytes injected with 10 ng of β2_α4_β2_α4_β2 cRNA (left/right panel) or 5 ng of β2_α4_β2_α4_α4 cRNA (right/left panel). For comparison the ACh EC₁₀₀ current responses of the corresponding non-linked receptors are included. Arrows indicate the start of the application of ACh.

Figure 4

Functional sensitivity of concatenated and non-linked (α4)₂(β2)₃ and (α4)₃(β2)₂ nAChR to α4β2-preferring ligands. Concentration-response curves were obtained for the effects of A85380, 5I-A8530, TC2559 and DhβE on (A) β2_α4_β2_α4_β2 and (B) β2_α4_β2_α4_α4 nAChR expressed heterologously in Xenopus oocytes as described in Methods. Averaged parameters of best fits to agonist or concentration-response data are given in Tables 2 and 3. For comparison the concentration-response curve constructed for the corresponding non-linked receptors have been included in (A) and (B).

Figure 5

Zn²⁺ sensitivity and Ca²⁺ permeability of concatenated (α4)₂(β2)₃ and (α4)₃(β2)₂ nAChR expressed heterologously in Xenopus oocytes. (A) Averaged concentration-response for the effects of Zn²⁺ at concatenated (α4)₂(β2)₃ and (α4)₃(β2)₂ nAChRs. The effects of Zn²⁺ on currents activated by EC₂₀ or EC₁₀ ACh concentrations on concatenated and non-linked (α4)₂(β2)₃ and (α4)₃(β2)₂ nAChRs, respectively, were determined as detailed in the methods. (B) Current-voltage relationship of β2_α4_β2_α4_β2 and β2_α4_β2_α4_α4 nAChR in the presence of 1.8 or 18 mmol·L⁻¹ extracellular Ca²⁺. β2_α4_β2_α4_α4 were more permeable to Ca²⁺ as judged by the positive shift of the reversal potential when the external Ca²⁺ was increased by 10-fold.

Figure 6

Chaperone 14-3-3 increases functional expression of concatenated (α4)₂(β2)₃ and (α4)₃(β2)₂ nAChR. Bargraph of normalized ACh responses at concatenated (A) β2_α4_β2_α4_β2 or (B) β2_α4_β2_α4_α4 nAChR receptors expressed on their own or co-expressed with chaperone protein 14-3-3. Data are given as means ± SEM from five to seven oocytes per column. *P < 0.05 and ***P < 0.001, statistically significant difference from corresponding concatenated receptors expressed on their own (unpaired Student's t-test).