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.
Highlights
Synaptic transmission is terminated by rapid clearance of the neurotransmitter from the synaptic cleft
Amphetamine-induced Conformational Change in the N Terminus of serotonin transporter (SERT) Detected by Tryptic Digestion—In the alternating access model, the transport cycle of SERT is linked to a conformational cycle where the hydrophobic core switches from an outward to an inward facing conformation
Molecular dynamics simulations predict that the N terminus of DAT is flexible, engages PIP2, triggers the opening of the inner gate of via an interaction with intracellular loop 4, and promotes release of Naϩ from the Na2 site; these actions are causally related because the N terminus is directed to IL-4 by engaging PIP2 molecules [18]
Summary
Amphetamine-induced Conformational Change in the N Terminus of SERT Detected by Tryptic Digestion—In the alternating access model, the transport cycle of SERT is linked to a conformational cycle where the hydrophobic core switches from an outward to an inward facing conformation. Membranes were prepared from HEK293 cells stably expressing wild type SERT tagged with a yellow fluorescent protein at the N terminus (YFP-human SERT (hSERT)) and incubated in the presence of 150 mM NaCl or of 150 mM choline chloride, conditions known to promote the outward facing and the inward facing conformation, respectively. Forward and Reverse Transport by N-terminal SERT Truncation Variants—If the sequences of the N termini of NET, DAT, and SERT are aligned, it is not possible to identify a any conserved element other than a highly conserved stretch of amino acids (RETWGKK; delineated in italics in Fig. 2a) preceding the first transmembrane helix (Fig. 2a) 182.1 Ϯ 35.0 181.3 Ϯ 24.6 203.4 Ϯ 11.3 86.8 Ϯ 26.7a a p Ͻ 0.001, significantly different from control
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