(A) In this and other recordings, a 17–20 hr treatment with nicotine (Nic, 10 μM) induced a robust, ~5 fold, increase in peak current amplitudes evoked by 1 mM ACh from α4β2R-expressing HEK cells. Varenicline (Var, 30 μM) treatment for an equivalent time had no effect on ACh-evoked peak current amplitudes alone but prevented upregulation of nAChR function when co-incubated with nicotine. Traces represent currents evoked by ACh in cells that were untreated or treated for 17 hr with vehicle, nicotine, varenicline, or with varenicline and nicotine. The number of recordings were 16, 18, 18, and 17, respectively. (B) Chronic lobeline (Lob, 30 μM) exposure reduced the peak ACh current amplitude relative to control and prevent nicotine upregulation. Traces represent currents evoked by ACh in cells that were untreated or treated for 17 hr with vehicle, nicotine, lobeline, or with lobeline and nicotine. The number of recordings were 13, 18, 13, and 15, respectively. (C) NH4Cl treatment (two 10 min washes) partially reverses the suppressive effects of varenicline on nicotine-induced functional upregulation. Traces show representative currents evoked by ACh from cells pretreated for 17–20 hr with vehicle, nicotine, or nicotine and varenicline before exposure to NH4Cl. The varenicline-alone condition is omitted for clarity. The graph is as in (A) except that the cells were treated with NH4Cl as noted. The profound reduction in nicotine-induced upregulation caused by varenicline co-incubation was lessened following treatment with NH4Cl. The number of recordings were 21, 22, 25, and 25, respectively. (D) Traces show representative currents evoked by ACh from cells pretreated for 17–20 hr with vehicle, nicotine, or nicotine and lobeline before exposure to NH4Cl. In this dataset, lobeline-induced suppression of nicotine upregulation was modestly attenuated by treatment with NH4Cl. The number of recordings were 14, 15, 16, and 19, respectively. In all the column graphs: * p<0.05; **p<0.01; ***p<0.001 by one-way ANOVA with Tukey’s multiple comparison test. Columns show group mean and error bars are the s.e.m.