Amphetamine activates calcium channels through dopamine transporter-mediated depolarization

Abstract

Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation.

Keywords: Excitability; L-type Ca(2+) channels; MDMA; Monoamine transporters; Neurotransmitter transport; Serotonin; Stimulants.

Figure 1. S(+)AMPH is more potent than DA as an hDAT substrate

A. Confocal images show hDAT expression in Flp-hDAT cells (red stain in bottom panel) but not in parental Flp-In^Tm T-REx^TM cells (top panel). DAPI nuclear staining is depicted in blue. Flp-hDAT cells show specific [³H] DA uptake with the following fitting parameters (see Eq. 1): EC₅₀ = 2.73 ± 0.49 μM, Hill slope = 1.8 ± 0.5 (n = 18). B. Competition of [³H]DA uptake using cold dopamine or S(+)AMPH yielded the following fitting parameters (Eq. 1): IC₅₀ = 12.05 ± 1.79 μM, Hill Slope = 0.8 ± 0.1 (n = 9) and IC₅₀ = 0.98*** ± 0.12 μM, Hill slope = 1.1 ± 0.1 (n = 11, ***p < 0.001 vs. IC₅₀ DA competition, t-test) for DA and S(+)AMPH, respectively.

Figure 2. S(+)AMPH or DA activates Ca_V1.2 and Ca_V1.3, but not Ca_V2.2

(Upper and middle panel) Intracellular Ca²⁺ determinations in Fura-2AM loaded Flp-hDAT cells evaluated by fluorescence microcopy, under constant perfusion and at 35°C. Flp-hDAT cells were co-transfected with Ca_V1.3, Ca_V1.2 or Ca_V2.2 plus β₃, α₂δ and EGFP plasmids. The α1 subunit was omitted from the plasmid transfection mix for the control condition. Transfected cells were identified by their EGFP signal and then briefly exposed to dopamine 10 μM (DA), S(+)AMPH 5 μM, high potassium external solution 130 mM (K⁺, equimolar substitution of Na⁺) or 4Br-A23187 (calcium ionophore, 5 μM) as indicated in the timeline of each panel. Isradipine (2 μM) averts Ca²⁺ signals induced by both hDAT substrates. Each trace constitutes the mean ± s.e.m. of n ≥ 81 cells per condition. (Lower panel) Voltage dependence of Ca_V1.2, Ca_V1.3 and Ca_V2.2- mediated Ca²⁺ currents: HEK293T cells were co-transfected with β3, α2δ, and EGFP expression plasmids plus alternatively Ca_V1.3, Ca_V1.2 or Ca_V2.2 plasmids. The Ca²⁺ current (I_Ca) recordings were carried out at room temperature under constant perfusion. Test pulses in 5 mV steps for Ca_V2.2 or 10 mV steps for Ca_V1.2 and Ca_V1.3 were applied from a holding potential of −80 mV. Representative responses are shown for Ca_V1.3 (light grey circle), Ca_V1.2 (dark grey triangle) and Ca_V2.2 (black square) and the magnitude of the test potentials are indicated in mV. The peak current density for the voltage steps were fit to Eq. 2 and yielded the following parameters: G_max = 497 ± 86, 560 ± 128 and 631 ± 77 (pS/pF); V_1/2 = −25.6 ± 1.0, −3.2 ± 0.8 and 5.5 ± 1.0 mV (***p < 0.001, one-way ANOVA, indicated in the figure); k = 6.7 ± 0.2, 7.7 ± 0.2 and 4.7 ± 0.1 (mV) for Ca_V1.3 (n = 8), Ca_V1.2 (n = 7) and Ca_V2.2 (n = 8), respectively.

Figure 3. hDAT-mediated depolarization activates L-type Ca²⁺ channels in Flp-hDAT cells

Intracellular Ca²⁺ concentration was determined by fluorescence microscopy in Flp-hDAT or the parental Flp-In^TM T-REx^TM 293 (Flp-In) cells (no hDAT expression) cells co-transfected with Ca_V1.2 (A, C, E) or Ca_V1.3 (B, D,F) plus β₃, α₂δ and EGFP plasmids, using the Ca²⁺ sensitive dye Fura-2AM. The experiments were carried out under constant perfusion at 35°C. The transfected cells were identified by their EGFP signal. A, B. Cells were briefly exposed to DA 10 μM, S(+)AMPH 5 μM or high potassium external solution 130 mM (K⁺) as indicated in each panel. C, D. The blockade of hDAT with methylenedioxypyrovalerone (MDPV, 1 μM) prevented Ca²⁺ signals induced by hDAT substrates. E, F. Cells were exposed to external solution containing Li⁺ (equimolar substitution of Na⁺) or external solution with high potassium as indicated in the timeline of each panel. Li⁺-induced Ca²⁺ signals only take place in cells expressing hDAT. Traces represent the mean ± s.e.m. of n ≥ 30 cells per condition.

Figure 4. S(+)AMPH is more potent than DA producing Ca²⁺ signals in L-type Ca²⁺ channel- expressing Flp-hDAT cells

Intracellular Ca²⁺ signals were monitored using the calcium sensitive dye Fura-2AM and epifluorescence microscopy. Three days prior to each experiment Flp-hDAT cells were co-transfected with Ca_V1.3 or Ca_V1.2 plus β₃, α₂δ and EGFP plasmids. The EGFP was used as transfection marker. (A, B, C, D) The potency of each compound was calculated using a two-pulse protocol, where one fixed saturating concentration pulse of dopamine works as an internal calibration and a variable concentration pulse was applied to get a dose-response curve. The traces represent the mean ± s.e.m. of n ≥ 48 cells per concentration. E. The dose-response curve was obtained fitting the responses to the Eq.1; cells expressing Ca_V1.3 the EC₅₀ values and Hill slope were: 693 ± 25 and 102*** ±16 nM (***p < 0.001 one way ANOVA) and 2.5 ± 0.2 and 0.9*** ± 0.1 (***p < 0.001 one way ANOVA) for dopamine and S(+)amphetamine, respectively. Cells expressing Ca_V1.2 the EC₅₀ values and Hill slope were: 916 ± 54 and 144*** ± 11 nM (***p < 0.001 one way ANOVA) and 1.7 ± 0.2 and 1.2 ± 0.1 for DA and S(+)AMPH, respectively, and (F) for better comparison the EC₅₀ values are plotted.

Figure 5. S(+)AMPH-induced currents are electrically favored to activate L-type Ca²⁺ channels

Ionic currents were determined by voltage-clamp in whole cell configuration under constant perfusion at 35°C. (A and B) Flp-hDAT cells clamped at −60 mV were exposed to a constant DA calibration pulse and a variable pulse of DA or S(+)AMPH; representative traces for the indicated concentrations tested are depicted in each panel. C. The full dose-response curves were fit to Eq.1 and yield the following parameters: EC₅₀ = 1.44 ± 0.24 μM and 0.28** ± 0.04 μM (**p < 0.01, t-test) and Hill Slope = 1.1 ± 0.1 and 1.0 ± 0.1 for DA and S(+)AMPH, respectively. Each point indicates mean ± s.e.m. of n ≥ 5 current determinations for each concentration. D. The fitted S(+)amphetamine and dopamine Ca²⁺ signals on Fig. 4E were plotted as a function of the fitted hDAT currents and (E) the coupling-strength index was computed as the first derivative of the Ca²⁺ signal – hDAT current curves.