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Papers In Press, published online ahead of print March 9, 2006
Pharmacology Dept., University of Vienna, Vienna A-1090
Corresponding Author: michael.freissmuth{at}meduniwien.ac.at
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 the exterior of the cell, respectively. It is not clear, how the conformational switch is triggered in neurotransmitter:sodium symporters (NSS), but Na+ is likely to play an essential role. Here, we focused on E136 of the serotonin transporter (SERT); this residue is conserved in transmembrane segment 2 of NSS and related proteins. Three substitutions were introduced resulting in SERT-E136D, SERT-E136Q, SERT-E136A, which were all correctly inserted into the plasma membrane. SERT-E136Q and SERT-E136A failed to support substrate influx into cells, while SERT-E136D did so at a reduced rate. Binding experiments with the inhibitor [3H]-
J. Biol. Chem, 10.1074/jbc.M511382200
Submitted on October 19, 2005
Revised on March 9, 2006
Accepted on March 9, 2006
The conserved glutamate (E136) in TM2 of the serotonin transporter is required for the conformational switch in the transport cycle
CIT supported the conjecture that the mutated transporters preferentially adopted the inward facing conformation: [3H]-CIT interacted with SERT in a manner consistent with binding to the outward facing state. Accordingly, the Na+-induced acceleration of [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, while SERT-E136A was inactive. Thus, in the absence of E136, the conformational equilibrium of SERT is shifted progressively (SERT-E136D > SERT-E136Q > SERT-E136A) to the inward facing conformation.
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