Using Ca2+-channel biosensors to profile amphetamines and cathinones at monoamine transporters: electro-engineering cells to detect potential new psychoactive substances

Abstract

Background-The appearance of stimulant-class new psychoactive substances (NPS) is a frequent and significant problem in our society. Cathinone variants are often sold illegally as 3,4methylenedioxymethamphetamine ("ecstasy") or disguised for legal sale using misleading names such as "bath salts" and carry the risk of promoting disruptive mental states, addiction, and fatal overdose. The principal targets of these recreational drugs are monoamine transporters expressed in catecholaminergic and serotonergic neurons. Some transporter ligands can be transported into cells, where they can promote a massive release of neurotransmitters through reverse transport, and others can block uptake. A ligand's dopamine vs. serotonin transporter selectivity, potency, and activity as a substrate or blocker can help elucidate the abuse liability and subjective effects of a drug.Objectives-Here we describe the discovery, development, and validation of an emerging methodology for compound activity assessment at monoamine transporters. Key findings-Substrates generate inward electrical currents through transporters, and can depolarize the plasma membrane, whereas blockers work as a "cork in a bottle" and f unction as antagonists. Voltage-gated Ca 2+ channels were co-expressed with monoamine transporters in cultured cells and used to measure fluctuations of the membrane electrical potential. In this system, substrates of monoamine transporters produce reliable dose-dependent Ca 2+ signals while blockers hinder them. Discussion-This system constitutes a novel use of voltage-gated Ca 2+ channels as biosensors for the purpose of characterizing ligand activity at monoamine transporters using fluorimetry. This approach in combination with in vivo evaluations of drugs' abuse-related effects is a powerful strategy for anticipating potential stimulant-class NPS.

Synthetic Cathinone Analogues Structurally Related to the Central Stimulant Methylphenidate as Dopamine Reuptake Inhibitors

“…It is well-accepted that substrate transport through DAT is associated with inward electrical currents that depolarize the plasma membrane . Uptake inhibitors, on the other hand, although they interact with the transporter, cannot induce the structural transitions that activate this depolarizing conductance. , As shown previously, voltage-gated Ca 2+ channels are excellent sensors of membrane depolarization that can be used as sensitive biosensors to detect substrate activity at DAT. , In this experimental setting, substrates of DAT (e.g., DA) produce fast and reversible Ca 2+ signals that can be measured using fluorescence microscopy, and inhibitors do not produce such responses but can interfere with the substrate-induced signal if applied together (Figure ). None of the compounds (i.e., 4 – 11 ) produced Ca 2+ signals when perfused alone, strongly suggesting that all are uptake inhibitors (data not shown).…”

“…19,20 As shown previously, voltage-gated Ca 2+ channels are excellent sensors of membrane depolarization that can be used as sensitive biosensors to detect substrate activity at DAT. 18,21 In this experimental setting, substrates of DAT (e.g., DA) produce fast and reversible Ca 2+ signals that can be measured using fluorescence microscopy, and inhibitors do not produce such responses but can interfere with the substrate-induced signal if applied together (Figure 2). None of the compounds (i.e., 4−11) produced Ca 2+ signals when perfused alone, strongly suggesting that all are uptake inhibitors (data not shown).…”

Methcathinone decreases dopamine transporter function: Role of protein kinase C

“…The amphetamine‐like drug METH increases PKC activity within the striatum through a calcium‐dependent pathway (Giambalvo, 2003), and intracellular calcium chelation with EGTA has been shown to attenuate METH‐induced decrease in plasmalemmal [ 3 H]DA uptake in vitro (Pubill et al., 2005). Given that S(‐)MCAT has been shown to produce significant increases in intracellular calcium (Steele & Eltit, 2019), the role of intracellular calcium was investigated. The MCAT‐induced decrease in plasmalemmal [ 3 H]DA uptake was attenuated by co‐incubation with the intracellular calcium chelator, BAPTA‐AM.…”

Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats

“…31,32 The amplitude of this Ca 2+ signal is proportional to the concentration of monoamine transporter substrate applied during the experiment, and by using several concentrations of substrate together with intracellular Ca 2+ determinations, dose−response curves can be constructed to estimate the potencies of substrates at transporters. 15,30−33 This methodology has been described in detail before; 34 briefly, stable and inducible cell lines expressing DAT, SERT, or NET, all of human origin, were previously made using the targeted recombination system HEK293 Flp-In T-REx (Invitrogen) following the manufacturer's suggested procedure. 31,32 Cells were plated in 96-well imaging plates and were cotransfected with plasmids encoding voltage-gated Ca 2+ channels (rabbit α1.2, rat β3, rat α2δ) for cells expressing DAT and SERT or (rat α1.3, rat β3, rat α2δ) for cells expressing NET.…”

“…The transport of substrates happens concomitant with inward currents depolarizing the cell membrane. This depolarization activates the voltage-gated Ca 2+ channels producing an intracellular Ca 2+ signal. , The amplitude of this Ca 2+ signal is proportional to the concentration of monoamine transporter substrate applied during the experiment, and by using several concentrations of substrate together with intracellular Ca 2+ determinations, dose–response curves can be constructed to estimate the potencies of substrates at transporters. ,− This methodology has been described in detail before; briefly, stable and inducible cell lines expressing DAT, SERT, or NET, all of human origin, were previously made using the targeted recombination system HEK293 Flp-In T-REx (Invitrogen) following the manufacturer’s suggested procedure. , Cells were plated in 96-well imaging plates and were cotransfected with plasmids encoding voltage-gated Ca 2+ channels (rabbit α1.2, rat β3, rat α2δ) for cells expressing DAT and SERT or (rat α1.3, rat β3, rat α2δ) for cells expressing NET. Although using α1.2 or α1.3 does not alter the apparent potency of the tested compound in this functional assay, α1.3 facilitates signal acquisition in cells expressing NET (empirical observation).…”