The conserved glutamate (Glu136) in transmembrane domain 2 of the serotonin transporter is required for the conformational switch in the transport cycle.
The alternate access model provides the theoretical framework for understanding how transporters translocate hydrophilic substrates across the lipid bilayer. The model postulates at least two conformations of a transporter, an outward and an inward facing conformation, which seal the translocation pathway to the interior and exterior of the cell, respectively. It is not clear how the conformational switch is triggered in neurotransmitter/sodium symporters, but Na+ is likely to play an essential role. Here, we focused on Glu136 of the serotonin transporter (SERT); this residue is conserved in transmembrane domain 2 of neurotransmitter/sodium symporters and related proteins. Three substitutions were introduced, resulting in SERT-E136D, SERT-E136Q, and SERT-E136A, which were all correctly inserted into the plasma membrane. SERT-E136Q and SERT-E136A failed to support substrate influx into cells, whereas SERT-E136D did so at a reduced rate. Binding experiments with the inhibitor 2beta-[3H]carbomethoxy-3beta-(4-iodophenyl)tropane (beta-[3H]CIT) supported the conjecture that the mutant transporters preferentially adopted the inward facing conformation: beta-[3H]CIT interacted with SERT in a manner consistent with binding to the outward facing state. Accordingly, the Na+-induced acceleration of beta-[3H]CIT association was most pronounced in wild-type SERT, followed by SERT-E136D > SERT-E136Q > SERT-E136A. Similarly, SERT-E136Q supported substrate efflux in a manner indistinguishable from wild-type SERT, whereas SERT-E136A was inactive. Thus, in the absence of Glu136, the conformational equilibrium of SERT is shifted progressively (SERT-E136D > SERT-E136Q > SERT-E136A) to the inward facing conformation.
Korkhov
Vladimir M
VM
Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
Holy
Marion
M
Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
Freissmuth
Michael
M
Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria. Electronic address: michael.freissmuth@meduniwien.ac.at.
Sitte
Harald H
HH
Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
D019943:Amino Acid Substitution; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D005786:Gene Expression Regulation; D005973:Glutamine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism
D050486:Serotonin Plasma Membrane Transport Proteins; D005973:Glutamine; D012964:Sodium; D002794:Choline; D011188:Potassium