We observed a significant decrease of current amplitudes at higher concentrations of α5, and this effect was significantly more pronounced with α5 D397N. These results suggest that α5 and β4 may compete for binding to α3, in line with the studies showing such competition for binding to α4 ( Gahring and Rogers, 2010). Given that overexpression of β2 with either α3 (Figure 1A) or α4 (Figure S1B) did not increase currents,

we were interested in identifying the residues differing between β4 and β2 that mediate this effect. Since the long cytoplasmic loop is the most divergent domain between nAChR subunits (Figure S1C), and since it has been implicated in cell-surface expression and trafficking of β2 subunits (Nashmi et al., 2003 and Ren et al., 2005), we generated β2–β4 chimeras exchanging either this domain, or short motifs Protein Tyrosine Kinase inhibitor and single residues within this domain. Replacement of the cytoplasmic loop of β2 with the corresponding sequences present in β4 (β2/β4 322–496) led to strong increase of nicotinic currents (Figure 1C). Introduction of two β4-specific motifs (a serine/tyrosine rich motif [β2/+β4 382–391] and gephyrin-like-binding motif [β2/+β4 401–419] into the β2 loop) had no influence on current amplitudes (Figure 1C). We next performed bioinformatic analyses and 3-Methyladenine nmr singled out eight β4-specific residues (indicated as T-1 to T-8 in Figure S1C) present

within highly conserved motifs. Six of these residues were not

further considered: T-2, T-3, T-6, and T-7 residues differ between mouse and chicken β4 subunits, which are equally potent in enhancing nicotine-evoked currents (Figure S1B); T-4 residue lies within the tested motif in the β2/+β4 382–391 chimera; and residues at position T-8 have the same charge (Figure S1C). The remaining two candidates, T-1 (S324 in β4 and T327 in β2) and T-7 (S435 in β4 and R431 in β2) (Figure S1C) were tested by point mutagenesis in the β2 subunit backbone. The β2 T327S point mutant did not increase current, whereas replacement of β2 R431 with serine resulted in a 3.5-fold current increase (Figure 1C). Furthermore, ever point mutation of the native S435 in the β4 subunit to the arginine residue present in β2 (β4 S435R) abolished the β4-specific activity. Thus, these data demonstrate that the distinctive ability of β4 to increase currents when overexpressed maps to a single residue (S435) that is required in β4 for current increase and can confer this property to β2. Alignment of mouse, human, and Torpedo nAChR subunit sequences indicated that S435 in β4 and D397N in α5 are located in the 25 amino-acid-long amphipathic membrane-associated stretch (MA-stretch) described in the Torpedo subunits ( Unwin, 2005) ( Figure 2A). Electron microscopy studies of the Torpedo nAChR have proposed a 3D density map of the receptor complex.