Amphetamine activates calcium channels through dopamine transporter-mediated depolarization
Graphical abstract
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)
- et al.
Mechanisms of neurotransmitter release by amphetamines: a review
Prog. Neurobiol.
(2005) - et al.
Conducting states of a mammalian serotonin transporter
Neuron
(1994) - et al.
Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation
Biophys. J.
(1996) - et al.
Channels in transporters
Curr. Opin. Neurobiol.
(1996) Regulating the conducting states of a mammalian serotonin transporter
Neuron
(2003)- et al.
Dopamine transport currents are promoted from curiosity to physiology
Trends Neurosci.
(2003) - et al.
Electrical coupling between the human serotonin transporter and voltage-gated Ca(2+) channels
Cell Calcium
(2014) - et al.
Ca(V)1 and Ca(V)2 channels engage distinct modes of Ca(2+) signaling to control CREB-dependent gene expression
Cell
(2012) - et al.
Differential expression of the noradrenaline transporter in adrenergic chromaffin cells, ganglion cells and nerve fibres of the rat adrenal medulla
J. Chem. Neuroanat.
(2001) - et al.
Distribution of calcium channel Ca(V)1.3 immunoreactivity in the rat spinal cord and brain stem
Neuroscience
(2009)