title: The N Terminus Specifies the Switch between Transport Modes of the Human Serotonin Transporter.
abstract: The serotonin transporter (SERT) and other monoamine transporters operate in either a forward transport mode where the transporter undergoes a full transport cycle or an exchange mode where the transporter seesaws through half-cycles. Amphetamines trigger the exchange mode, leading to substrate efflux. This efflux was proposed to rely on the N terminus, which was suggested to adopt different conformations in the inward facing, outward facing and amphetamine-bound states. This prediction was verified by tryptic digestion of SERT-expressing membranes: in the absence of Na+, the N terminus was rapidly digested. Amphetamine conferred protection against cleavage, suggesting a relay between the conformational states of the hydrophobic core and the N terminus. We searched for a candidate segment that supported the conformational switch by serial truncation removing 22 (ΔN22), 32 (ΔN32), or 42 (ΔN42) N-terminal residues. This did not affect surface expression, inhibitor binding, and substrate influx. However, amphetamine-induced efflux by SERT-ΔN32 or SERT-ΔN42 (but not by SERT-ΔN22) was markedly diminished. We examined the individual steps in the transport cycle by recording transporter-associated currents: the recovery rate of capacitive peak, but not of steady state, currents was significantly lower for SERT-ΔN32 than that of wild type SERT and SERT-ΔN22. Thus, the exchange mode of SERT-ΔN32 was selectively impaired. Our observations show that the N terminus affords the switch between transport modes. The findings are consistent with a model where the N terminus acts as a lever to support amphetamine-induced efflux by SERT.
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria.
authors.forename: Carina
authors.initials: C
authors.lastname: Kern
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria.
authors.forename: Fatma Asli
authors.initials: FA
authors.lastname: Erdem
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria.
authors.forename: Ali
authors.initials: A
authors.lastname: El-Kasaby
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Sandtner
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, 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 of Vienna, A-1090 Vienna, Austria sonja.sucic@meduniwien.ac.at.
authors.forename: Sonja
authors.initials: S
authors.lastname: Sucic
mesh_terms: D000662:Amphetamines / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D019921:Biotinylation; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020128:Inhibitory Concentration 50; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D018377:Neurotransmitter Agents / Q000737:chemistry; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D000072417:Protein Domains; D012701:Serotonin / Q000737:chemistry; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry*; D012964:Sodium / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000662:Amphetamines; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D018377:Neurotransmitter Agents; D050486:Serotonin Plasma Membrane Transport Proteins; C054562:yellow fluorescent protein, Bacteria; D012701:Serotonin; D012964:Sodium; D014357:Trypsin
keywords: neurotransmitter release; neurotransmitter transport; patch clamp; serotonin; serotonin transporter