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.

Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl- Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na+, and Cl- in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl- on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstrate ions and substrate such that their forward transport mode was maintained even under conditions of elevated intracellular Na+ or Cl- The most important differences in the transport cycle of GlyT1 and GlyT2 arose from the kinetics of charge movement and the resulting voltage-dependent rate-limiting reactions: the kinetics of GlyT1 were governed by transition of the substrate-bound transporter from outward- to inward-facing conformations, whereas the kinetics of GlyT2 were governed by Na+ binding (or a related conformational change). Kinetic modeling showed that the kinetics of GlyT1 are ideally suited for supplying the extracellular glycine levels required for NMDA receptor activation.

Phosphatidylinositol-4,5-bisphosphate (PIP2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2 . Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors.