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J Biol Chem. 2016 Dec 9;291(50):25864-25876. Epub 2016 Oct 18.

Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter.

Author information

1
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
2
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria walter.sandtner@meduniwien.ac.at.

Abstract

The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na+ concentrations because intracellular Na+ release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K+ and, notably, also H+ In addition, our data suggest that Cl- is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K+ or of H+ to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na+ release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na+ release and K+ (or H+) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form.

KEYWORDS:

electrophysiology; kinetics; monoamine transporter; neurotransmitter transport; serotonin

PMID:
27756841
PMCID:
PMC5207061
DOI:
10.1074/jbc.M116.753319
[Indexed for MEDLINE]
Free PMC Article

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