External cysteine residues in the serotonin transporter

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• The rat serotonin transporter (SERT) is a key target for antidepressants.
• Cysteine residues in membrane proteins can be critical for structure and function.
• Extracellular cysteines in SERT are potential sites for functional modification.

Purpose of the Study:

• To investigate the roles of three predicted extracellular cysteines (C109, C200, C209) in SERT function and expression.
• To determine the accessibility and potential interactions of these cysteine residues.

Main Methods:

• Site-directed mutagenesis of SERT cysteine residues to alanine or serine.
• Expression of wild-type and mutant SERT in HeLa cells.
• Functional assays measuring transport activity.
• Surface expression analysis.
• Chemical modification using methanethiosulfonate (MTS) reagents.

Main Results:

• C109 mutation had no effect on transport or expression; its accessibility is cation-dependent.
• Mutations at C200 and C209 significantly reduced transport activity and surface expression.
• C200S mutant showed increased sensitivity to MTS reagents, suggesting altered accessibility.
• Double mutants C200S-C109A and C200S-C209S retained partial activity.
• C200 and C209 mutations affected Na+ dependence of transport and ligand binding.
• C200S-C209S double mutant showed normal surface expression, unlike single mutants.

Conclusions:

• C200 and C209 are essential for SERT's transport activity and proper surface expression.
• C200 and C209 may be linked by a disulfide bond in the second extracellular loop of SERT.
• C109's accessibility to external reagents is modulated by cation binding.