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.

Serotonin transport in the 21st century

“…The relationship of the peak current to voltage indicates the valence, or the amount of charge transferred across the membrane. The voltage dependence of the peak current measured in the nominal absence of intracellular Na + was consistent with slightly more than one full charge moving through the membrane electric field upon conversion to an inward-facing conformation and dissociation of Na + (Hasenhuetl et al, 2016). We found that raising cytoplasmic Na + decreased the current, consistent with the essential involvement of Na + dissociation in this process (Fig.…”

“…Electroneutrality of the overall reaction implies that an equal and opposite charge movement must occur somewhere else in the cycle. The peak current also allowed us to confirm that the turnover rate of SERT is voltage independent (this has been verified for voltages ranging from −80 mV to +50 mV), reinforcing observations that the rate-determining steps are unlikely to move charge across the membrane (Hasenhuetl et al, 2016). Additional study of this process allowed us to determine the rates for most of the partial reactions for this transporter operating in the forward direction (Schicker et al, 2012;Hasenhuetl et al, 2018).…”

“…In both cases (internal K + or low internal pH), recovery is insensitive to membrane potential, indicating that the steps following K + or H + binding do not move charge across the membrane. Additionally, low internal pH, like internal K + , decreased the amount of charge crossing the membrane with the peak current, consistent with a site that could bind K + or H + as Na + dissociates (Hasenhuetl et al, 2016). These results explain how a transmembrane pH difference (internally acidic) could drive 5-HT accumulation (Keyes and Rudnick, 1982).…”

“…Although neither intracellular 5-HT nor Cl − affected the charge moving in the peak current, increasing intracellular K + decreased the peak current almost as much as Na + (Fig. 9 E) and similarly decreased the valence of the peak current (Hasenhuetl et al, 2016). This suggested that K + binding to inward-open SERT was a voltage-dependent process, possibly involving conformational changes that compensate electrically for the entry of charge with Na + .…”

Functional Selectivity and Partial Efficacy at the Monoamine Transporters: A Unified Model of Allosteric Modulation and Amphetamine-Induced Substrate Release

“…The two phenomena (uptake vs. release) are subject to two different rate-limiting reactions, which are kinetically distinct: monoamine uptake is rate limited by the slow return of the empty transporter to the outward-facing state (Bulling et al, 2012;Schicker et al, 2012;Hasenhuetl et al, 2016); monoamine efflux is rate limited by intracellular substrate exchange and subsequent outward translocation of the corresponding monoamine (Hasenhuetl et al, 2018). Hence, relative preference to bind either the substrateloaded or substrate-free transporter (i.e., conformational selection) affects kinetically distinct partial reactions and can afford functional selectivity (Li et al, 2017).…”