title: Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter.
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.
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Hasenhuetl
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.affiliation: 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.
authors.forename: Walter
authors.initials: W
authors.lastname: Sandtner
mesh_terms: D002414:Cations, Monovalent / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D011522:Protons; D012701:Serotonin / Q000235:genetics / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
chemical_list: D002414:Cations, Monovalent; D011522:Protons; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D012964:Sodium; D011188:Potassium
keywords: electrophysiology; kinetics; monoamine transporter; neurotransmitter transport; serotonin