Publications

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

  Hasenhuetl P.S., Freissmuth M., Sandtner W.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 291:25864-25876(2016) [PubMed] [EuropePMC]

• Serotonin transport in the 21st century.

  Rudnick G., Sandtner W.

  Serotonin (5-hydroxytryptamine [5-HT]) is accumulated within nerve endings by the serotonin transporter (SERT), which terminates its extracellular action and provides cytoplasmic 5-HT for refilling of synaptic vesicles. SERT is the target for many antidepressant medications as well as psychostimul ... >> More

  Serotonin (5-hydroxytryptamine [5-HT]) is accumulated within nerve endings by the serotonin transporter (SERT), which terminates its extracellular action and provides cytoplasmic 5-HT for refilling of synaptic vesicles. SERT is the target for many antidepressant medications as well as psychostimulants such as cocaine and ecstasy (3,4-methylenedioxymethamphetamine). SERT belongs to the SLC6 family of ion-coupled transporters and is structurally related to several other transporter families. SERT was studied in the 1970s and 1980s using membrane vesicles isolated from blood platelets. These studies led to a proposed stoichiometry of transport that has been challenged by high-resolution structures of SERT and its homologues and by studies of SERT electrophysiology. Here, we review the original evidence alongside more recent structural and electrophysiological evidence. A self-consistent picture emerges with surprising insights into the ion fluxes that accompany 5-HT transport. << Less

  J Gen Physiol 151:1248-1264(2019) [PubMed] [EuropePMC]

• Platelet 5-hydroxytryptamine transport, an electroneutral mechanism coupled to potassium.

  Rudnick G., Nelson P.J.

  Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. ... >> More

  Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. Addition of valinomycin induces a membrane potential of approximately 50 mV (interior negative) as estimated by uptake of the lipophilic cation triphenylmethylphosphonium, but has surprisingly little effect on 5-hydroxytryptamine transport. Addition of 2,4-dinitrophenol dissipates the valinomycin-induced membrane potential. In the absence of valinomycin, 2,4-dinitrophenol has no effect on 5-hydroxytryptamine transport but valinomycin and 2,4-dinitrophenol together inhibit transport, probably by dissipation of the K+ gradient. These results are consistent with an electroneutral mechanism in which 5-hydroxytryptamine influx is directly coupled to potassium ion efflux and argue against an electrogenic mechanism in which there is a net influx of positive charge with 5-hydroxytryptamine. << Less

  Biochemistry 17:4739-4742(1978) [PubMed] [EuropePMC]

• Coupling between platelet 5-hydroxytryptamine and potassium transport.

  Nelson P.J., Rudnick G.

  J Biol Chem 254:10084-10089(1979) [PubMed] [EuropePMC]

• The serotonin transporter-imipramine "receptor".

  Talvenheimo J., Fishkes H., Nelson P.J., Rudnick G.

  The platelet plasma membrane serotonin transporter requires Na+ for two reactions, serotonin transport and imipramine binding. Although imipramine binding has been thought to reflect the same process required for serotonin binding prior to transport (Talvenheimo, J., Nelson, P.J., and Rudnick, G. ... >> More

  The platelet plasma membrane serotonin transporter requires Na+ for two reactions, serotonin transport and imipramine binding. Although imipramine binding has been thought to reflect the same process required for serotonin binding prior to transport (Talvenheimo, J., Nelson, P.J., and Rudnick, G. (1979) J. Biol. Chem. 254, 4631-4635), binding and transport display markedly different responses to Na+. Imipramine binding (and competitive inhibition of transport) apparently requires two sodium ions which bind with a KD of 300 +/-70 meq/liter. The total number of sites (Bmax) is the same at all Na+ concentrations, but the affinity for imipramine increases from 7.3 x 10(6) M-1 at 20 meq/liter to 110 x 10(6) M-1 at 200 meq/liter. Na+ acts, at least in part, by decreasing the rate of imipramine dissociation from its binding site. Serotonin binding displaces imipramine from its site on the membrane. In contrast to imipramine binding, this displacement is a simple, hyperbolic function of Na+ concentration with a KD for Na+ of 400 +/-100 meq/liter, which suggests that only one Na+ is required. Serotonin transport is also much less responsive to Na+ concentration. Over the same concentration range in which the affinity for imipramine increases 15-fold, the affinity for serotonin increases only 2-fold. Despite the lack of Na+ effect on the Bmax for imipramine binding, the Vmax for serotonin transport increases as a simple saturable function of Na+ with a KM (Na+) of 52 meq/liter. Thus, substrate translocation as well as binding requires Na+. Since serotonin is cotransported with Na+, the serotonin gradient accumulated depends on the coupling stoichiometry and the magnitude of the Na+ gradient imposed. From the response of the serotonin gradient to imposed Na+ gradients, we calculated a serotonin:Na+ cotransport stoichiometry of 0.9. Taken together, the results suggest that serotonin and imipramine bind either to the same site or to mutually exclusive sites, but maximal imipramine binding requires two sodium ions, while maximal serotonin binding and translocation requires only one. << Less

  J Biol Chem 258:6115-6119(1983) [PubMed] [EuropePMC]