Effect of nicotine, ER export, and retention/retrieval motifs on nAChR expression at the PM. (A) Representative TIRFM images of Neuro-2a cells transfected with α4eGFP β2wt and α4eGFP β2-365AAQA368 L349M double-mutant receptors. Column labels indicate the expressed receptors. Panels indicate the nicotine treatment condition. Bars, 10 µm. Each image is accompanied by a graph plotting the fluorescence intensity (x axis) versus number of pixels (y axis) for whole TIRF footprint (ER + PM) and ER. (B) Bar graph quantifying the PM-integrated density for α4eGFP plus β2wt and α4eGFP plus β2 double-mutant–transfected cells. Transfected β2 subunits and nicotine treatment conditions are indicated below the x axis. Error bars show 99% confidence intervals. (C) Integrated density ratios of whole TIRF footprint to ER for α4eGFP β2wt and α4eGFP β2 double-mutant nAChRs. The transfected β2 subunit and nicotine treatment conditions are indicated below the x axis; error bars are SEM. The number of imaged cells is indicated in parentheses. (D; 1 and 2) Protein sequence alignments of ER export and retention motifs within the M3–M4 loop of β2 and β4 subunits. ER export motifs are in green, and ER retention/retrieval motifs are in red. 1, protein sequence comparison for mouse β2 and β4 subunits; 2, cross-species comparison of the M3–M4 cytoplasmic loop sequences for mouse, human, and chicken β2 nAChR subunits. All β2 sequences include amino acids 321–382, and the β4 sequence includes amino acids 318–378.