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 Ca2+ channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca2+ 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 Ca2+ channels (CaV1.3 and CaV1.2) but not the N-type Ca2+ 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 Ca2+ 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 Ca2+ channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca2+ channel activation.

Effects of N-Alkyl-4-Methylamphetamine Optical Isomers on Plasma Membrane Monoamine Transporters and Abuse-Related Behavior

“…21–23 In our calcium fluorescence assay, substrate-induced transporter-mediated currents are coupled to voltage-gated Ca 2+ channels which causes opening of calcium channels increasing the intracellular Ca 2+ concentration. 15,24,25 As we have described before 15 and as shown here, cells expressing these two proteins and loaded with a cytosolic Ca 2+ sensor constitute a biosensor that produces a detectable Ca 2+ signal in response to transporter-mediated currents induced by substrates (Figure. S3A).…”

“…To improve the dynamic range of these recordings the experiments where repeated using a different Ca 2+ channel, Ca V 1.3, that has a lower threshold of activation and requires less depolarization to open. 25 When Ca V 1.3 rather than Ca V 1.2 was used, the efficacy of 4 S and 4 R to induce Ca 2+ signals increased to 88% and 68%, respectively. The improvement of the signal, when Ca V 1.3 was used, suggests that 4 S and 4 R act as partially efficacious substrates to evoke modest inward current at NET.…”

“…15,24,25 Briefly, stable cell lines expressing each transporter were developed using the FlpIn-TREx system (Invitrogen). Cells were plated in 96-well flat-bottom imaging plates, then were co-transfected with plasmids coding α 1 , β 3 , α 2 γ and EGFP in a ratio (1:0.5:1:0.2, in μg) using Fugene 6 as transfecting reagent.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…The actions of AMPH to elevate Ca 2+ levels were blocked by desipramine, suggesting that AMPH-induced depolarization may be responsible for Ca 2+ channel activation. Consistent with this idea, Cameron et al (2015) recently reported an ability of AMPH to activate VGCCs via transporter-mediated depolarization. Although these studies were of DAT expressing cells, NET is also electrogenic and generates significant currents in response to substrate interactions (Galli et al, 1998).…”

Structure-Activity Relationships of Substituted Cathinones, with Transporter Binding, Uptake, and Release

“…These potentially depolarizing transporter-mediated substrate-induced sodium currents (Sonders et al, 1997;Sitte et al, 1998) cause reverse transport or release of neurotransmitter. The exogenous substrate-induced sodium currents can put cells at risk, as (1)fenfluramine is associated with neuronal depletion of 5-HT , and amphetamine can indirectly activate voltage-gated Ca 21 channels (Cameron et al, 2015). The substrates identified here, especially those that had efficacies similar to METH, may cause depolarization in neurons, as evidenced by the dose-dependent currents elicited by 4-MEC in Xenopus oocytes expressing hSERT (Saha et al, 2015).…”