Heterologous expression of concatenated nicotine acetylcholine receptors: pros and cons of subunit concatenation and recommendations for construct designs.

Concatenation of Cys-loop receptor subunits is a commonly used technique to ensure experimental control of receptor assembly. However, we recently demonstrated that widely used constructs did not lead to the expression of uniform pools of ternary and more complex receptors. The aim was therefore to identify viable strategies for designing concatenated constructs that would allow strict control of resultant receptor pools.Concatenated dimeric, tetrameric and pentameric α4β2-containing nACh receptor constructs were designed with successively shorter linker lengths and expressed in Xenopus laevis oocytes. Resulting receptor stoichiometries were investigated by functional analysis in two-electrode voltage clamp experiments. Molecular dynamics simulations were performed to investigate potential effect of linkers on the 3D-structure of concatemers.Dimeric constructs were found to be unreliable and should be avoided for expression of ternary receptors. By introducing two short linkers, we obtained efficient expression of uniform receptor pools with tetrameric and pentameric constructs. Yet, linkers should not be excessively short as that introduces strain on the 3D-structure of concatemers.The data demonstrate that design of concatenated Cys-loop receptors requires a compromise between the desire for control of assembly and avoiding introduction of strain on the resulting protein. The overall best strategy was found to be pentameric constructs with carefully optimised linker lengths. Our findings will significantly facilitate studies of ternary or more complex Cys-loop receptors as well as enabling detailed analysis of how pharmacological agents interact with stoichiometry-specific binding sites.