FIGURE 2.

The α5 subunit enters into stable complexes with α4 and up-regulation to nicotine is reduced. 293 cells were co-transfected with varying amounts of α5 cDNA (as indicated) with constant cDNA amounts for either α4 and β2 or α4 and β4, respectively. A, immunoprecipitation with either anti-α5 or the appropriate anti-β subunit was followed by Western blot analysis to determine the amount of associated α4. Quantitation of the blots in this panel shows that as the amount of α5 cDNA co-transfected with α4+β2 subunit is increased, associations between α4 and both β2 and α5, respectively, also increase. In contrast, when β4 is substituted for β2, the amount of α4 associated with β4 diminishes, whereas α5 is enhanced. All of the values were normalized to the no α5 input values, and the error bars were calculated and are shown as ± S.E. for the results from three to seven independent experiments. B, [³H]EB ligand binding to membranes from cells transfected as in A and membranes from cells treated with 1 μm nicotine (gray bars). Increased α5 corresponds with decreased up-regulation induced by nicotine (fold increase is noted above each treatment set). Each experiment was conducted three to eight times, and the error bars reflect ± S.E. for all experiments.