Figure 3.

[³H]Dopamine ([³H]DA) uptake in HEK293 cells expressing the hDAT-PG^584,585AA after preincubation with ibogaine analogs. (A) Comparison of [³H]DA uptake in HEK293 cells transiently expressing WT hDAT or hDAT-PG^584,585AA. Each symbols represents the result from an individual experiment (done in triplicate); means ± SD are also shown. The statistical comparison was done via a Mann–Whitney test (p < 0.0001). (B) Cells expressing hDAT-PG^584,585AA were incubated in the presence of the indicated compounds (30 μM in all instances but bupropion, 100 μM). After 24 h, specific [³H]DA uptake was determined as outlined in the “Materials and Methods” section. Values from individual experiments (done in triplicate) are shown as dots; box plots show the median and the interquartile range. The statistical comparison was done by one-way ANOVA with a post hoc Dunnett’s multiple comparison to confirm a statistically significant difference between untreated control cells and those exposed to individual compounds (*, p < 0.05; ***, p < 0.001). (C, top) Concentration–response curves for pharmacochaperoning by the indicated compounds (selected as positive hits from panel B). Rescued uptake was normalized to that achieved by 30 μM noribogaine (= 100%) to account for inter-experimental variations. Rescue by 9b was plotted separately because of its very high efficacy in comparison to those of other analogs (C, bottom). The solid lines were drawn by fitting the data to the equation for a rectangular hyperbola (for EC₅₀ and E_max of mutant protein rescue, see Table 1). Data were obtained in at least three independent experiments carried out in triplicate. The error bars indicate SD.