Structural Determinants of α4β2 Nicotinic Acetylcholine Receptor Trafficking

• DOI: 10.1523/jneurosci.1079-05.2005
• Publication Date: 2005-07-14T18:59:09Z
• Authors: Xiao-Qin Ren; Shi-Bin Cheng; Magdalen W. Treuil; Jayanta Mukherjee; Jayaraman Rao; K. H. Braunewell; Jon M. Lindstrom; Rene Anand
• Keywords: Neurons; 0301 basic medicine; Pyridines; Cell Membrane; Molecular Sequence Data; Bridged Bicyclo Compounds, Heterocyclic; Endoplasmic Reticulum; Kidney; Cell Line; Protein Structure, Tertiary; Rats; Neuroblastoma; Protein Transport; 03 medical and health sciences; Amino Acid Substitution; Leucine; Mutagenesis, Site-Directed; Animals; Humans; Amino Acid Sequence; Protein Multimerization; Hydrophobic and Hydrophilic Interactions

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 α4β2 AChR subunits were required for cell surface expression of α4β2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the α4 AChR subunit attenuates cell surface expression of mutant α4β2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the β2 AChR subunit abolishes cell surface expression of mutant α4β2 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 β2 AChR subunit only poorly inhibits trafficking of mutant α4β2 AChR to the cell surface. All mutant α4β2 AChRs exhibit high-affinity binding for [3H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type α4β2 AChRs colocalize with the Golgi marker giantin, whereas mutant α4β2 AChRs fail to do so. The striking difference between mutant α4 versus mutant β2 AChR subunits on cell surface expression of mutant α4β2 AChRs points to a cooperative or regulatory role for the α4 AChR subunit and an obligatory role for the β2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of α4β2 AChR ER export.