Elsevier

Cell Calcium

Volume 58, Issue 5, November 2015, Pages 457-466
Cell Calcium

Amphetamine activates calcium channels through dopamine transporter-mediated depolarization

https://doi.org/10.1016/j.ceca.2015.06.013Get rights and content

Highlights

  • Amphetamine (AMPH) and dopamine (DA) induce depolarizing currents through the DA transporter (DAT).

  • DAT substrate-induced depolarization activates L-type but not N-type Ca2+ channels (CaV).

  • AMPH is more potent than DA when activating L-type CaV.

  • The activation of L-type CaV displays higher coupling-strength with AMPH- than DA-induced currents.

  • AMPH action likely involves L-type CaV activation.

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.

Introduction

The dopamine transporter (DAT) is a Na+/Cl-dependent symporter expressed in dopaminergic neurons; its principal function is to limit dopamine receptor signaling by restricting the extracellular concentration of dopamine (DA) [1], [2]. Amphetamine (AMPH) is a DAT substrate and its more potent enantiomer, S(+)AMPH, is used therapeutically to treat attention deficit hyperactivity disorder and narcolepsy [2], [3]. AMPH competes with and diminishes DA uptake. In addition, intracellular AMPH disrupts DA's internal stores and induces the reverse transport of DA through DAT, increasing extracellular DA concentration [4], [5]. Accordingly, the activation of dopaminergic pathways in the brain accounts for both the therapeutic properties and addictive liability of AMPH and its active derivatives [2], [6], [7].

An additional level of complexity for AMPH's action in cells is the generation of DAT-mediated, AMPH-induced inward currents [8], [9], [10], [11]. Although substrate-induced currents through monoamine transporters are widely accepted [12], [13], [14], [15] and they have been implicated in neurotransmitter depletion in the brain [16], the physiological significance of such currents are still under debate [17], [18]. Recently, we showed that depolarization induced by serotonin (5HT) or S(+)3,4-methylenedioxymethamphetamine (MDMA, ecstasy) in skeletal muscle cells engineered to express the human serotonin transporter (hSERT) activates the L-type Ca2+ channel CaV1.1 [19]. Similarly, hSERT-mediated depolarization activates the L-type Ca2+ channel CaV1.3 in HEK cells, whereas hSERT activation is unable to open the N-type Ca2+ channel CaV2.2 under identical experimental conditions [19]. The L-type Ca2+ channels are important modulators of signal transduction and excitability in excitable cells. In particular, CaV1.3 and CaV1.2 have been extensively studied upstream of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cAMP response element-binding protein (CREB) signaling pathways in neurons [20], [21], [22]. Furthermore, the lower-threshold L-type CaV1.3 channel is implicated in pace-making in dopaminergic neurons, and in neuroendocrine cells, such as adrenal chromaffin cells [23], [24]. Since L-type channels CaV1.2 and CaV1.3 are expressed with monoamine transporters in several excitable cells [23], [24], [25], [26], [27], [28], [29], determining a functional interaction between these two classes of proteins could constitute an additional molecular mechanism of AMPH action.

In the present study we co-expressed the human DAT (hDAT) with CaV1.2, CaV1.3 or CaV2.2 in Flp-In™ T-REx™ 293 cells, and measured the effect of S(+)AMPH- or DA-induced DAT currents on CaV activation. These experiments were designed to study the interplay between two variables: (1) the affinity of S(+)AMPH and DA on hDAT, and (2) the voltage sensitivity of the Ca2+ channels studied, in achieving effective hDAT-CaV coupling. The results show that, regardless of the compound affinity on hDAT, DA and S(+)AMPH can couple indirectly to both L-type channels (CaV1.2 and CaV1.3) but not to the N-type channel (CaV2.2) under identical conditions. In addition, whereas the potency to achieve hDAT-CaV electrical coupling is dominated by substrate-hDAT affinity, the coupling-strength, defined as the Ca2+ signal change per unit hDAT current, is influenced by the sensitivity of Ca2+ channels to voltage. Moreover, S(+)AMPH showed larger coupling-strength compared to DA at concentrations that induced relatively small hDAT-mediated currents. These results suggest that S(+)AMPH- and DA-induced currents through hDAT are qualitatively different, because the S(+)AMPH-induced current is pharmacologically and electrically stronger at activating L-type channels.

Section snippets

Generation of Flp-In™ T-REx™ cells expressing the human dopamine transporter (Flp-hDAT cells) and CaV channel transfection

The generation of the hDAT stable inducible cell line (Flp-hDAT) was done using the Flp-In™ T-REx™ 293 system (Life Technologies). The hDAT cDNA (accession number: NM_001044) was subcloned into the pcDNA5/FRT/TO plasmid and the targeted single site recombination and cell selection were performed as described previously [19]. The Ca2+ channels used in this study were CaV2.2 (α1B, Addgene #26570), CaV1.3 (α1D, Addgene #26576), CaV1.2 (α1C accession number: NM_001136522), β3 (Addgene #26574) and

Results

Immunostaining in conjunction with confocal microscopy showed membrane localization of hDAT in Flp-hDAT cells three days after doxycycline induction, whereas the parental Flp-In™ T-REx™ 293 (Flp-In) cells showed no hDAT expression (insert, Fig. 1A). In addition, Flp-hDAT cells have specific [3H]DA uptake (EC50 = 2.73 ± 0.49 μM, Fig. 1A). Uptake competition assay using cold S(+)AMPH or cold DA yielded inhibition constants (Ki) equal to 0.24 ± 0.03 and 2.06*** ± 0.67 μM respectively (*** = p < 0.001 t-test n  

Discussion

Voltage-gated Ca2+ channels are composed of the main α1 subunit and the auxiliary α2δ, β and γ subunits [37]. The α1 subunit contributes to the ionic pore and voltage sensor structures, while the others modulate expression, targeting, and function [33], [38], [39]. Neurons and neuroendocrine cells express several α1 isoforms. The biophysical properties, location and biochemical partners of the α1 subunits regulate Ca2+ influx for specific purposes. For example, the opening of L-type Ca2+

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors would like to thank Dr. Richard A. Glennon (Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA) for insightful discussions and for providing methylenedioxypyrovalerone used in this work. This study was supported by the National Institute of Health R01 DA033930 (L.J.D.).

References (53)

  • S. Schroeter et al.

    Polarized expression of the antidepressant-sensitive serotonin transporter in epinephrine-synthesizing chromaffin cells of the rat adrenal gland

    Mol. Cell. Neurosci.

    (1997)
  • E.A. Ertel et al.

    Nomenclature of voltage-gated calcium channels

    Neuron

    (2000)
  • L. Borre et al.

    The second sodium site in the dopamine transporter controls cation permeation and is regulated by chloride

    J. Biol. Chem.

    (2014)
  • J. Szpyt et al.

    Three-dimensional localization of the alpha and beta subunits and of the II-III loop in the skeletal muscle L-type Ca2+ channel

    J. Biol. Chem.

    (2012)
  • B.A. Simms et al.

    Neuronal voltage-gated calcium channels: structure, function, and dysfunction

    Neuron

    (2014)
  • A. Vaarmann et al.

    Novel pathway for an old neurotransmitter: dopamine-induced neuronal calcium signalling via receptor-independent mechanisms

    Cell Calcium

    (2010)
  • J.U. Fog et al.

    Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport

    Neuron

    (2006)
  • T. Steinkellner et al.

    Ca(2+)/calmodulin-dependent protein kinase IIalpha (alphaCaMKII) controls the activity of the dopamine transporter: implications for Angelman syndrome

    J. Biol. Chem.

    (2012)
  • J.S. Goodwin et al.

    Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in vivo

    J. Biol. Chem.

    (2009)
  • K. Schicker et al.

    Unifying concept of serotonin transporter-associated currents

    J. Biol. Chem.

    (2012)
  • B. Giros et al.

    Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter

    Nature

    (1996)
  • P. Seeman et al.

    Anti-hyperactivity medication: methylphenidate and amphetamine

    Mol. Psychiatry

    (1998)
  • E. Mignot

    Perspectives in narcolepsy research and therapy

    Curr. Opin. Pulm. Med.

    (1996)
  • A.E. Fleckenstein et al.

    New insights into the mechanism of action of amphetamines

    Annu. Rev. Pharmacol. Toxicol.

    (2007)
  • N.D. Volkow et al.

    Dopamine in drug abuse and addiction: results from imaging studies and treatment implications

    Mol. Psychiatry

    (2004)
  • N.D. Volkow et al.

    Dopamine in drug abuse and addiction: results of imaging studies and treatment implications

    Arch. Neurol.

    (2007)
  • Cited by (0)

    View full text