期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 292, 期 9, 页码 3603-3613出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.771360
关键词
neurotransmitter release; neurotransmitter transport; patch clamp; serotonin; serotonin transporter
资金
- Austrian Science Fund/FWF [P27518-B27, P28090, F3510]
- Austrian Science Fund (FWF) [P28090, P27518] Funding Source: Austrian Science Fund (FWF)
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 (N22), 32 (N32), or 42 (N42) N-terminal residues. This did not affect surface expression, inhibitor binding, and substrate influx. However, amphetamine-induced efflux by SERT-N32 or SERT-N42 (but not by SERT-N22) 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-N32 than that of wild type SERT and SERT-N22. Thus, the exchange mode of SERT-N32 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.
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