Equimolar amounts of plasmids coding for dimeric and monomeric constructs were used. Cells plated in a 6-well plate at 70% of confluency were transfected using 3 mg of cDNAs and 9 ml of jetPEI (DNA/jetPEI ratio 1:3) per well. After 24 h at 37°C, cells were lysed or fixed.

The alpha3beta4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 beta4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the beta4 and alpha3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 beta4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of beta4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the beta4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in alpha3beta4 nicotinic receptor is more than an accessory subunit.