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


METHODS
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.


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
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.
Carbone
A-L
AL
School of Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford, UK.
Moroni
M
M
Groot-Kormelink
P-J
PJ
Bermudez
I
I
D048948:14-3-3 Proteins / Q000096:biosynthesis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D044708:DNA, Concatenated / Q000235:genetics; D066298:In Vitro Techniques; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D015202:Protein Engineering; D055503:Protein Multimerization; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation; D014982:Xenopus laevis; D017967:Zinc Compounds / Q000494:pharmacology
D048948:14-3-3 Proteins; D002712:Chlorides; D044708:DNA, Concatenated; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D017967:Zinc Compounds; C016837:zinc chloride; D002118:Calcium