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The dopamine, serotonin, and norepinephrine transporters are important determinants of monoaminergic neurotransmission. The pharmacological manipulation of these systems contributes not only to drug addiction, but also to the treatment of many mental diseases. The development of novel tools to expedite the identification of drug-effect profiles on monoamine transporters can benefit efforts to discover new psychotherapeutics.Monoamine transporters are natively expressed in the plasma membranes of presynaptic neurons, where they mediate the uptake of monoamine neurotransmitters. When expressed in heterologous expression systems, the uptake of substrates is associated with an inward current that depolarizes the cell membrane. In addition, voltage-gated Ca2+ channels modulate Ca2+ permeability as a function of the membrane potential. We took advantage of these two elemental principles to develop a new assay to detect and characterize ligands of monoamine transporters.Using the Flp-In T-REx expression system, we established permanent cell lines expressing the human dopamine or serotonin transporters. We studied the ability of these cells to produce Ca2+ signals in response to monoamine transporters’ substrates after transient transfection with different voltage-gated Ca2+ channels. In addition we studied the dynamic range and sensitivity of two genetically encoded Ca2+ sensors. Our results show that the co-expression of CaV1.2 and GCamp6s in monoamine transporter-expressing cells generates significant Ca2+ signals in response to monoamine transporter substrates. Moreover monoamine transporter blockers can inhibit these Ca2+ signals. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Supported by R01 DA033930 and R01 AR067738. |
