FIGURE 6.

HSP inhibitors synergize with the pharmacochaperone ibogaine in rescuing the folding-deficient SERT mutants SERT-F604Q (A) and SERT-R607A/I608A (B) but not SERT-P601A/G602A (C). A–C, HEK293 cells stably expressing YFP-tagged SERT-F604Q (F604Q; A), SERT-R607A/I608A (RI-AA; B), and SERT-P601A/G602A (PG-AA; C) seeded onto 48-well plates and incubated in the absence (Control; closed squares) and presence of 17-DMAG (2 μm; closed triangles) or pifithrin-μ (5 μm; open squares) with the indicated concentrations of noribogaine for 24 h. Cell surface expression of the transporters was quantified by measuring specific [³H]5-HT uptake. Data are from three to four independent experiments done in triplicate; error bars indicate S.E. The curves were generated by fitting the data points to the equation of a rectangular hyperbola. D, detergent lysates were prepared from HEK293 cells expressing YFP-tagged SERT-P601A/G602A, which had been incubated in the absence (Control lane) and presence of noribogaine (10 μm), pifithrin-μ (5 μm), a combination of noribogaine and pifithrin-μ, 17-DMAG (2 μm), and a combination of noribogaine and 17-DMAG for 24 h. After electrophoretic separation and transfer to nitrocellulose membranes, immunoreactivity of SERT was detected via its N-terminal tag with an antibody directed against GFP (upper blot). C and M indicate the position of the core glycosylated (ER-resident) and mature (Golgi and post-Golgi) forms of SERT, respectively. The blot was also probed with an antibody recognizing Gβ subunits as a loading control (lower blot). The bar diagram represents the densitometric quantification of the mature fully glycosylated (M) corrected for loading. The intensity seen under control conditions was set to 1, and the -fold change observed in the presence of compounds was averaged from three independent experiments (means ± S.E.).