Jensen_2013_J.Biol.Chem_288_33708

Explorations into the alpha6-containing nicotinic acetylcholine receptors (alpha6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric alpha6/alpha3 and mutant alpha3 and alpha6 subunits and functional characterization of these co-expressed with beta4 or beta4beta3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of alpha6 with the corresponding alpha3 segment or residue was found to enhance alpha6beta4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both alpha3 elements into alpha6 resulted in assembly of alpha6beta4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by alpha6beta4beta3 receptors comprising one of these novel alpha6/alpha3 chimeras in oocytes were found to be in good agreement with those from previous studies of alpha6* nAChRs formed from other surrogate alpha6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this alpha6/alpha3 chimera with beta2 or beta2beta3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in alpha6 could be specific impediments for the expression of functional alpha6beta4* nAChRs.