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Accessibility and Conformational Coupling in Serotonin Transporter Predicted Internal Domains

Overview
Journal J Neurosci
Specialty Neurology
Date 2002 Sep 28
PMID 12351711
Citations 21
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Abstract

The intracellular topology of serotonin transporter (SERT) was examined using mutants containing single cysteine residues in the predicted cytoplasmic domain of the protein. Cysteine residues in each predicted cytoplasmic domain, including the NH2 and COOH termini and the five predicted internal loops, reacted with methanethiosulfonate (MTS) reagents only when the plasma membrane was permeabilized with digitonin or in membrane preparations but not in intact cells. The reaction was monitored by inactivation of high-affinity binding activity and by incorporation of biotin groups into the protein. Of the seven endogenous cysteine residues predicted to lie in the cytoplasmic domain, modification of only Cys-357 in the third internal loop (IL3) led to loss of activity. Cys-15 in the NH2 terminus and Cys-622 in the COOH terminus also reacted with MTS reagents. Modification of cysteine residues inserted at positions 137 in IL1, 277 in IL2, and 441 in IL4 also led to inactivation, and at positions 157 in IL1 and 532 in IL5, cysteine was modified without an effect on binding activity. These results are in agreement with the originally proposed topology for SERT and argue against an alternative topology proposed for the closely related GABA and glycine transporters. The reactivity of many of the cytoplasmic cysteine residues studied was influenced by ion and ligand binding, suggesting that the internal domains of SERT participate in conformational changes during neurotransmitter transport.

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References
1.
Chen J, Rudnick G . Permeation and gating residues in serotonin transporter. Proc Natl Acad Sci U S A. 2000; 97(3):1044-9. PMC: 15515. DOI: 10.1073/pnas.97.3.1044. View

2.
Kilic F, Rudnick G . Oligomerization of serotonin transporter and its functional consequences. Proc Natl Acad Sci U S A. 2000; 97(7):3106-11. PMC: 16200. DOI: 10.1073/pnas.97.7.3106. View

3.
Ni Y, Chen J, Androutsellis-Theotokis A, Huang C, Moczydlowski E, Rudnick G . A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109. J Biol Chem. 2001; 276(33):30942-7. DOI: 10.1074/jbc.M104653200. View

4.
Androutsellis-Theotokis A, Ghassemi F, Rudnick G . A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter. J Biol Chem. 2001; 276(49):45933-8. DOI: 10.1074/jbc.M107462200. View

5.
Blakely R, Clark J, Rudnick G, Amara S . Vaccinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters. Anal Biochem. 1991; 194(2):302-8. DOI: 10.1016/0003-2697(91)90233-j. View