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Fluorescent substrates to study the human serotonin transporter in single-cells: a comparison between app+ and asp+ Ernesto Solís, Jr

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Ernesto Solís, Jr., Igor Zdravkovic, Ian D. Tomlinson, Louis J. DeFelice

Monoamine transporters terminate neurotransmission at the synapse and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening, but until now they have focused on catecholamine transporters, while their use for serotonin transporters (SERTs) has been less successful. Here we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare two fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP+) as reporters for the human serotonin transporter (hSERT) in single cells. A novel fluorescent substrate for hSERT, 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+), is superior to other MPP+ analogs exhibiting lower fluorescence accumulation in hSERT-expressing HEK293 cells. APP+ accumulation is biphasic, with a fast accumulation phase (km = 2.29  0.65 M) followed by a slower accumulation phase (km = 2.36  0.55 M). Na+- and Cl--dependent APP+ accumulation is displaced by serotonin and inhibited by fluoxetine, suggesting APP+ is suitable to monitor hSERT activity. However, APP+ does not display the fast fluorescent binding phase at hSERT that is observed with ASP+ in hNET. We compared APP+ to 4-(4-diethylaminostyryl)-N-methylpyridinium (ASP+), which was previously used to study the human norepinephrine transporter (hNET). ASP+ is 10 times less potent than APP+ at inhibiting serotonin uptake (ki = 180.1  20.3 M versus 19.7  2.2 M) and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped at -60 mV, APP+ induced hSERT-mediated inward currents (indicating APP+ is a substrate), whereas ASP+ induced hSERT-mediated outward currents, suggesting restricted uptake and activity as an inhibitor. Ligand-receptor docking of APP+ and ASP+ in an hSERT homology model showed ASP+ docked more tightly within the active region than APP+, providing confirmation of the fluorescent functional differences. We conclude APP+ is better suited than ASP+ to study hSERT transport fluorometrically.  

molecular stent hypothesis

Ernesto Solís, Jr., Aldo A. Rodriguez-Menchaca, Krasnodara Cameron, and Louis J. De Felice

Background and purpose:  Wherever they are located, dopamine transporters (DATs) clear dopamine (DA) from the extracellular milieu to help regulate dopaminergic signaling. Exposure to amphetamine (AMPH) increases extracellular DA in the synaptic cleft, which prolongs postsynaptic activity and reinforces abuse and hedonic behavior. The mechanisms behind AMPH-induced DA release are only partially understood, and AMPH-induced channels in DAT may play a hitherto unsuspected role. Key results and conclusions:  We have discovered a new action of S(+)AMPH on the human dopamine transporter (hDAT) that potentially relates to AMPH-induced DA release mechanisms. At -60mV, near the resting potential of neurons, S(+)AMPH induces a depolarizing current through hDAT; it has not been reported, however, that after removing S(+)AMPH a fraction of the initial current persists for up to 30 minutes. This is in contrast to R(-)AMPH-  and DA-induced currents, which return to baseline immediately after their removal. The initial S(+)AMPH-induced current is carried by S(+)AMPH and likely Na+, whereas the persistent current is carried by Na+ and possibly Cl-. The persistent current likely results from an internal action of S(+)AMPH on hDAT, because the time required for the effect to manifest is consistent with S(+)AMPH transport into the cell, and because intracellular injection of S(+)AMPH rapidly activates the persistent current. Remarkably, S(+)AMPH injection also activates a DA-induced persistent current. Experimental approach:  Xenopus laevis oocytes were injected with the hDAT cRNA and after several days were voltage-clamped and exposed to DA, R(-)AMPH, or S(+)AMPH. Implications:  We speculate that the persistent current requires S(+)AMPH interactions with an internal gate on hDAT, acting as a molecular stent that keeps the transporter open. Amphetamine-induced persistent currents may have major implications for dopaminergic signaling, DA release mechanisms, and amphetamine abuse. 

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