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.
Highlights
In the forward-transport mode, the transporter releases its substrate and co-substrates into the cytosol and completes the catalytic cycle by returning from the inward-facing conformation in an empty state or may countertransport other co-substrates (e.g. Kϩ)
The currents through serotonin transporter (SERT) have been reconciled with a kinetic model of its catalytic cycle [13]
Monoamine transporters can operate in both a forward transport and a substrate-exchange mode [3]; in the substrateexchange mode, the transporter returns from the inward-facing to the outward-facing conformation loaded with substrate and co-substrates
Summary
The transporter can enter a substrate-exchange mode; in this mode it switches between the substrate-bound outward-facing and the substrate-bound inward-facing conformations without completing its catalytic cycle. The transporter may translocate the extracellular substrate into the cell and return with an alternative substrate originating from the cytosol This results in release of the intracellular substrate. The transporter may subsequently return to the outwardfacing conformation loaded with the same substrate molecule because substrate dissociation from the inward-facing conformation is blocked. This precludes uptake and is physiologically undesirable. The switch between these transport modes and the key variables, which define this decision point, can only be understood, if intracellular (co-)substrate binding and release is addressed by time-resolved measurements.
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