Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression

Abstract

Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT after release. To explore protein kinase C-mediated SERT regulation, we generated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hSERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and antidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-cell currents. In these cells, the protein kinase C activator beta-PMA caused a time-dependent reduction in 5-HT uptake capacity (Vmax) after acute application and a reduction in SERT-mediated currents. Effects of beta-PMA were mimicked by the phorbol ester beta-PDBu, were not observed with the inactive alpha-isomers, and could be blocked by treatment of cells with the protein kinase C inhibitor staurosporine. Biotinylation/immunoblot analyses showed that activity reductions are paralleled by a staurosporine-sensitive loss of surface SERT protein. These data indicate that altered surface abundance, rather than reduced catalytic transport efficiency, mediates acute PKC-dependent modulation of 5-HT uptake.

Fig. 5.

β-PMA reduces SERT-mediated 5-HT currents in 293-hSERT cells. A, 293-hSERT cells were held at −40 mV and stepped to −120 mV for 500 msec with a 5 sec rest interval between pulses. After addition of 1 μm 5-HT, the current increased and reached a steady state in three or four steps. β-PMA (0.2 μm) added in the presence of 1 μm 5-HT always reduced the 5-HT-induced current by >75% within ∼5 min.B, hSERT-mediated currents from the cell recorded inA are revealed by subtraction of 5-HT-induced current from currents in the absence of 5-HT. C, The same subtraction procedure as in B after the addition of 0.2 μm β-PMA. In this experiment, β-PMA essentially abolished the 5-HT-induced current.

Fig. 6.

Staurosporine blocks the effect of β-PMA on 5-HT-induced currents. A, The protocol applied is that described in Figure 5. hSERT-mediated currents were induced by addition of (B) 1 μm 5-HT, and then (C) β-PMA (0.2 μm) was added in the presence of staurosporine (1 μm). Unlike the addition of β-PMA alone (Fig. 5), β-PMA plus staurosporine failed to reduce 5-HT-induced currents in all cells tested. In this experiment, staurosporine increased the 5-HT-induced current. The current seems to be hSERT-mediated, because 1 μm paroxetine reduced the 5-HT current by 80%.

Fig. 1.

Characterization of 293-hSERT cells.A, Immunoblot of 293-hSERT membranes with SERT antibody CT-2 (1 μg/ml). A single 96 kDa band is evident that is not present in parental HEK-293 cells. Each lane contains 50 μg of total protein extract. B, Equilibrium binding of [¹²⁵I]RTI-55 to 293-hSERT cell membranes. Membranes (7.5 μg) were incubated with various concentrations of RTI-55 for 1 hr at room temperature. Nonspecific binding was defined by incubations in the presence of 1 μm paroxetine, and data were subtracted from total bound to define specific binding. 293-hSERT cell membranes bind [¹²⁵I]RTI-55 with a K_D of 0.26 nm and a B_max of 1.2 pmol/mg protein, which, on the basis of membrane recovery, converts to ∼7 × 10⁴ sites/cell. The figure shown is a representative single binding isotherm, performed in triplicate, plotted ± SD. Two other replicates gave essentially equivalentK_D and B_maxvalues. C, 5-HT uptake by 293-hSERT cells. 293-hSERT cells exhibit saturable 5-HT uptake, with aV_max of 11.9 pmol/min/10⁶ cells, a K_M of 0.26 μm, and a Hill coefficient (n) of 1.6. Uptake was conducted in triplicate, plotted ± SD, and repeated once at this passage, although equivalent K_M values were obtained in five other experiments. Nonspecific uptake was defined as the uptake in the presence of 1 μm paroxetine and subtracted from total uptake. D, Antagonist sensitivity of 5-HT uptake in 293-hSERT cells. [³H]5-HT uptake (20 nm), assayed in triplicate and repeated with equivalent results, was inhibited by paroxetine (IC₅₀ = 0.15 nm), RTI55 (IC₅₀ = 1.7 nm), imipramine (IC₅₀ = 3.9 nm), citalopram (IC₅₀ = 7.4 nm), and cocaine (IC₅₀ = 0.3 μm). Nonspecific uptake was defined as the uptake in the presence of 1 μm of paroxetine (for RTI55, citalopram, imipramine, and cocaine inhibition) or 1 μm of RTI55 (for paroxetine inhibition) and subtracted from total uptake. Values are plotted as a percentage of specific 5-HT uptake obtained in the absence of antagonists and curve fit to a three-parameter logistic equation, % inhib = 100/(1 + (IC₅₀/[I]ⁿ).

Fig. 2.

5-HT-induced currents in 293-hSERT cells.A, Cells were clamped in whole-cell mode and stimulated with a 7 sec ramp from −120 mV to 20 mV before (control) or after addition of increasing concentrations of 5-HT to the bath. 5-HT-induced currents are not observed in nontransfected HEK-293 cells. Averaged data from experiments on five cells reveal a saturable 5-HT-induced current with a K_M = 0.21 ± 0.05 μmand n = 1.4 ± 0.2. B, Paroxetine (1 μm) blocks the 5-HT-induced current in 293-hSERT cells. Currents were evoked by 1 μm 5-HT as inA, before (control) or after application of paroxetine to the bath. In dose–response studies on five cells, paroxetine inhibited the 5-HT-induced current with aK_I of 1.4 ± 0.2 nm.

Fig. 3.

Regulation of 5-HT uptake in 293-hSERT cells by β-PMA. A, Time course of the inhibition of 5-HT uptake by β-PMA. Cells were preincubated with 1 μm β-PMA for the times indicated and then assayed for 5-HT (1 μm, 10 min) uptake, as described in Materials and Methods. Maximal inhibition of 5-HT uptake by β-PMA was observed after 30 min of pretreatment.B, Dose–response curve of β-PMA effects on 5-HT uptake in 293-hSERT cells. Cells were preincubated with various concentrations of β-PMA for 30 min, followed by 10 min 5-HT uptake assays. C, Kinetic analysis of the effect of β-PMA on 5-HT uptake in 293-hSERT cells. Cells were preincubated with 1 μm PMA or vehicle for 20 min before 10 min uptake assay at various concentrations of 5-HT, as indicated. Parallel assays were performed in the presence of 100 nm paroxetine to define specific 5-HT uptake. Velocities measured were normalized to transport rates at 1 μm to compensate for varying expression levels across passages (see Materials and Methods). β-PMA caused a 47% decrease of 5-HT transport V_max with little change in K_M (0.39 μm in control vs 0.28 μm in β-PMA-treated cells).Inset shows an Eadie–Hofstee transformation of the data. For A–C, data presented are the averaged data from three separate experiments performed with six replicates per concentration. *p < 0.05; **p< 0.01; two-sided Student’s t test.

Fig. 4.

Stereospecificity and staurosporine sensitivity of phorbol ester-induced reductions in 5-HT uptake. A, The effect of phorbol esters on 5-HT uptake in 293 cells is stereospecific. Cells were preincubated separately with α or β stereoisomers of PMA and PDBu for 30 min before uptake assays with [³H]5-HT (1 μm, 10 min). PKC-inactive α isomers were ineffective in reducing 5-HT uptake, unlike the β forms. Data presented are mean ± SEM of three experiments performed in triplicate.B, The effect of β-PMA on 5-HT uptake in 293-hSERT cells can be blocked by staurosporine. Cells were preincubated with 1 μm β-PMA, 1 μm staurosporine, or 1 μm β-PMA plus 1 μm staurosporine for 30 min. β-PMA (1 μm) significantly inhibited 5-HT uptake, but it became ineffective in the presence of 1 μmstaurosporine. Staurosporine alone (1 μm) had no effect on 5-HT uptake. Data presented are the mean ± SEM of three experiments performed in quadruplicate. Nonspecific uptake forA and B was defined as the uptake in the presence of 100 nm paroxetine and subtracted from total accumulation to yield specific uptake. *p < 0.05; **p < 0.01; two-sided Student’s ttest.

Fig. 7.

Effect of β-PMA on cell surface hSERT density. A, SERT immunoblot of total, nonbiotinylated, and biotinylated (cell surface) protein in 293-hSERT cells. Cells were treated with 1 μm β-PMA or vehicle for 40 min before biotinylation with sulfo-NHS-biotin. Aliquots (40 μl) of total, nonbiotinylated, and wash fractions were loaded, whereas the entire eluate (50 μl) from the streptavidin beads was loaded as the biotinylated sample and the blots were probed with CT-2 antibody (1 μg/ml). B, The same blot that was stripped and probed with anti-calnexin antibody to identify the endoplasmic reticular membrane protein calnexin. Calnexin immunoreactivity was observed only in the total and nonbiotinylated lanes and was used to normalize hSERT immunoreactivity between β-PMA-treated and untreated cells.C, Averaged quantitation of β-PMA effects on hSERT total, nonbiotinylated, and surface density. Immunoblots from three separate biotinylation experiments were scanned densitometrically, SERT values were normalized with calnexin immunoreactivity, and mean values were plotted ± SEM. Data are expressed as a percentage of vehicle-treated cells (control). Asterisk indicates a statistically significant reduction in biotinylated hSERT protein (p < 0.05; Student’s ttest). Two additional experiments to test staurosporine effects (Fig.8) gave consistent results, as did direct visualization of CT-2 immunoreactivity with [¹²⁵I]protein-A.

Fig. 8.

Staurosporine blocks β-PMA-induced reduction in hSERT surface abundance. A, Biotinylation experiments were performed with or without 1 μm β-PMA, as in Figure7, and blotted with CT-2 antibody. Separate wells were coincubated with 1 μm staurosporine. B, Calnexin immunoreactivity of the same blot as in A after stripping. C, Quantitation of the effect of staurosporine on total and biotinylated (cell surface) hSERT. CT-2 immunoreactive bands were scanned, and density values were normalized for calnexin immunoreactivity in the same cell extracts. Data are expressed as a percentage of values obtained with vehicle-treated cells (control). Staurosporine alone had little or no effect on total or surface hSERT protein, but its presence blocked the β-PMA-induced reduction in hSERT surface protein. The experiment was performed twice, yielding identical results.