Journal of Biological Chemistry
Volume 291, Issue 50, December 2016, Pages 25864-25876
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Molecular Biophysics
Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter*

https://doi.org/10.1074/jbc.M116.753319Get rights and content
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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.

electrophysiology
kinetics
monoamine transporter
neurotransmitter transport
serotonin

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*

This work was supported by the Austrian Science Fund/FWF Project P28090 (to W. S.), Project Program Grant SFB35 (F3510 to M. F.), and the MD/Ph.D. program of the Medical University of Vienna (to P. S. H.). This work is part of a dissertation submitted in partial fulfillment of the requirements of the Ph.D. degree. The authors declare that they have no conflicts of interest with the contents of this article.