Structural determinants of α4β2 nicotinic acetylcholine receptor trafficking

The structural determinants of nicotinic acetylcholine receptor ( AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum ( ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the alpha 4 beta 2 AChR subunits were required for cell surface expression of alpha 4 beta 2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the alpha 4 AChR subunit attenuates cell surface expression of mutant alpha 4 beta 2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the beta 2 AChR subunit abolishes cell surface expression of mutant alpha 4 beta 2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the beta 2 AChR subunit only poorly inhibits trafficking of mutant alpha 4 beta 2 AChR to the cell surface. All mutant alpha 4 beta 2 AChRs exhibit high-affinity binding for [H-3] epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type alpha 4 beta 2 AChRs colocalize with the Golgi marker giantin, whereas mutant alpha 4 beta 2 AChRs fail to do so. The striking difference between mutant alpha 4 versus mutant beta 2 AChR subunits on cell surface expression of mutant alpha 4 beta 2 AChRs points to a cooperative or regulatory role for the alpha 4 AChR subunit and an obligatory role for the beta 2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of alpha 4 beta 2 AChR ER export.