Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Abstract

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.

Keywords: amperometry; mass spectrometry; phosphoinositide; release; reuptake.

Fig. 1.

PIP₂ binding to SERT and regulation of SERT-mediated currents. (A) Lysates of either HEK293 cells expressing YFP-SERT (+) or nontransfected HEK293 (−) cells were incubated with either PIP₂-coated beads or control beads. Samples of these lysates or proteins eluted from these beads were separated by SDS/PAGE and immunoblotted with an anti-GFP antibody. (B–G) Current traces from single HEK293-SERT cells; application of pCA (3 µM) is indicated by the bar: traces were obtained in the presence (gray) and absence (black; Ctl) of 10 µM pal-HRQKHFEKRR (pal-peptide in B), 10 µM scrambled peptide (C), 10 µM m-3M3FBS (D), 10 µM o-3M3FBS (E), 10 µM m-3M3FBS plus 3µM U72133 (F), or 10 nM bradykinin (G). The peptides, m-3M3FBS, o-3M3FBS, m-3M3FBS plus U72133, or 10nM bradykinin had been present for 20 min before the currents were recorded. Cells in G were coexpressing B₂ bradykinin receptors. (Calibration bars, 2 pA and 2 s.)

Fig. 2.

PIP₂ regulation of SERT-mediated efflux. (A) Representative oxidative currents from HEK-SERT cells. d-Amph (10 µM) was added as indicated by the arrows. Traces were obtained from cells loaded with either 10 µM scrambled peptide (upper trace) or pal-HRQKHFEKRR. (B) Time course of pCA-induced, SERT-mediated efflux of [³H]MPP⁺ from HEK-SERT cells. The cells were loaded with 0.1 µCi [³H]MPP⁺ and superfused, and 2-min fractions were collected. The indicated concentrations of m-3M3FBS or 25 µM o-3M3FBS were added to the superfusion buffer at minute 4. At minute 14, pCA (3 µM) was added to induce efflux. Efflux of radioactivity per 2-min fraction is expressed as percentage of radioactivity present in the cells at the beginning of that fraction (n = 6; two-way ANOVA followed by Bonferroni's post hoc test: **P < 0.01 or ***P < 0.001). (C) Experiments were carried out as shown in B; 25 µM m-3M3FBS or o-3M3FBS, in the absence or presence of 3 µM U73122, was added at minute 4. Similarly, 30 µM PAO was compared against control. Bar graphs represent efflux in percent of total radioactivity between minutes 22 and 24 (n = 5–37; two-tailed Mann–Whitney test: **P < 0.01 or ***P < 0.001). (D) Experiments were carried out as shown in B; pCA-induced, SERT-mediated efflux of [³H]MPP⁺ from HEK-SERT cells coexpressing B₂ bradykinin receptors. Bradykinin (10 nM), either alone or together with 100 nM Hoe 140, was added to the superfusion buffer at minute 4. At minute 14, pCA (3 µM) was added to induce efflux (n = 3; one-way ANOVA followed by Bonferroni's post hoc test and is denoted by *P < 0.05). (E) Concentration dependence of m-3M3FBS treatment on the reduction of efflux in HEK293-DAT or HEK293-NET cells (n = 3; d-amphetamine, 3 µM). Cells were treated essentially as described in B. (F) Rat hippocampal slices were preloaded with 0.1 µM [³H]MPP⁺, and SERT-mediated efflux was induced by 3 µM pCA in the presence of m-3M3FBS or o-3M3FBS. Nomifensine (100 nM) was present throughout the experiment to block potential contributions from NET and DAT. Efflux is presented as in C–E. Statistically significant differences were assessed by Student t test (P < 0.05; n = 6, performed in triplicate).

Fig. 3.

Differences between SERT and GAT-1 in PIP₂ sensitivity. (A) Efflux of [³H]GABA from HEK-GAT1 cells. The cells were loaded with 0.2 µM [³H]GABA and superfused, and 2-min fractions were collected (as shown for SERT in Fig. 2); 25 µM of m-3M3FBS or o-3M3FBS was added to the superfusion buffer at minute 4. At minute 14, GABA (333 µM) was added to induce efflux. Bar graphs represent efflux in percent of total radioactivity between minutes 22 and 24 (n = 4). (B) Current traces from single HEK-GAT1 cells; application of GABA (100 µM) is indicated by the bar; traces were obtained in the presence (gray) and absence (black; Ctl) of 10 µM m-3M3FBS. (C) HEK-GAT1 cells in 24-well plates were preincubated in Krebs–Ringer–Hepes (KHB) buffer with the respective substances for 15 min before 0.2 µM [³H]GABA was added for 3 min; nonspecific uptake was measured in the presence of 10 µM tiagabine. Subsequently, the cells were washed with ice-cold buffer and lysed, and the cellular radioactive content was counted (n = 3). (D–F Electrostatic potential iso-surfaces of GAT1 (D), SERT (E), and SERT-R144A-K352A-K460A (F); red denotes negative potential and blue denotes positive potential. The colored ellipses point to the positions of residues R144 (red), K352 (green), and K460 (blue).

Fig. 4.

Lysine residues K352 and K460 in the cytosolic domain of SERT are required for binding and functional effects of PIP₂. (A–C) Time course of pCA-induced, SERT-mediated efflux of [³H]MPP⁺ from HEK293 cells expressing SERT-K352A, YFP-tagged SERT, or SERT-K352A-K460A. The cells were loaded with 0.1 µCi [³H]MPP⁺ and superfused, and 2-min fractions were collected; 25 µM m-3M3FBS or o-3M3FBS was added to the superfusion buffer at minute 4. At minute 14, pCA (3 µM) was added to induce efflux. Efflux of radioactivity per 2-min fraction is expressed as percentage of radioactivity present in the cells at the beginning of that fraction (n = 3–5). Statistical significance was calculated using two-way ANOVA followed by Bonferroni's post hoc test and is denoted by ***P < 0.001; n.s., no significance. (D) HEK293 cells stably expressing YFP-tagged SERT (WT) or SERT-K352A-K460A were solubilized (cell lysates) and pulled down with PIP₂-coated beads. Eluted proteins from the beads were dissolved in 2× SDS loading buffer at 95 °C, separated by SDS/PAGE, and immunoblotted with an anti-GFP antibody. The images were derived from the same membrane with the same exposure time and represent three experiments with the same results. (E) Uptake of [³H]MPP⁺ by HEK293 cells expressing SERT-K352A-K460A was essentially carried out as described in Table S1 (n = 3). (F) Inhibition of 5HT uptake by pCA. Cells expressing SERT-K352A-K460A or WT SERT were incubated with 0.03 µM [³H]MPP⁺ and increasing concentrations of pCA as indicated (n = 4); after 3 min, the cells were washed and lysed, and radioactivity was determined. The IC₅₀ values were 10.3 ± 0.9 and 3.7 ± 0.6 nM, respectively. (G) Current traces from single HEK293 cells expressing SERT-K352A-K460A (gray) or WT SERT (black); application of pCA (3 µM) is indicated by the bar.