Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 19, Pages 13439-13448Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M511382200
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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 Glu(136) 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 2 beta- [H-3] carbomethoxy- 3 beta- ( 4- iodophenyl) tropane (beta- [H-3] CIT) supported the conjecture that the mutant transporters preferentially adopted the inward facing conformation: beta- [H-3] CIT interacted with SERT in a manner consistent with binding to the outward facing state. Accordingly, the Na+- induced acceleration of beta- [H-3] CIT association was most pronounced in wildtype SERT, followed bySERT- 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.
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