Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

Melissa I Torres-Altoro,Charles P Kuntz,David E Nichols,Eric L Barker

doi:10.1074/jbc.m109.088138

Melissa I Torres-Altoro, Charles P Kuntz + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.088138

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Abstract

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT(Aa)) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln(332) in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln(332) to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.

Highlights

The serotonin (5-hydroxytryptamine (5-HT)2) transporter (SERT) is a member of the neurotransmitter sodium symporters (NSSs), whose function is to return 5-HT into presynaptic neurons after release into the synapse [1,2,3]
Construction and Transport Activity of human SERT (hSERT) Cys Mutant— The generation of a human serotonin transporter homology model based on the crystal structure of the A. aeolicus leucine transporter (LeuTAa), a bacterial homologue of the Naϩ/ClϪdependent transporters, provided us with the opportunity to develop a rational hypothesis for the identity of residues involved with substrate transport [5]
Residues modeled at the entrance to the permeation pore for hSERT were as follows: transmembrane ␣-helices (TMHs) I (Ile108), TMH VI (Ala330, Ala331, and Gln332), TMH X (Thr497 and Gly498), and extracellular loop 4 (EL4) (Glu396, Val397, and Ala398) (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Construction of hSERT Cys Mutants—The QuikChange௡ mutagenesis kit (Stratagene, La Jolla, CA) was used to generate the following hSERT Cys mutants: TMH I, I108C; TMH VI, A330C, A331C, and Q332C; TMH X, T497C and G498C; EL4, E396C, V397C, and A398C. Following MTSET treatment, cells were washed twice with PBS/CM, and the [3H]5-HT uptake assay was performed as described above. 48 h after transient transfection, HEK-293 cells (2 ϫ 104 cells/well) expressing the double mutant of interest were washed once with PBS/CM buffer and incubated with 100 ␮M MTS-3-MTS, 100 ␮M MTS-4-MTS, 100 ␮M propyl methanethiosulfonate (PMTS) (a monofunctional MTS reagent as a control), or buffer alone (total uptake) for 10 min at room temperature. Forty-eight hours after transient transfection with each mutant cDNA, the cells were washed twice with PBS and incubated with 300 ␮l of radioimmune precipitation solubilization buffer (10 mM Tris-base, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate) for 30 min while shaking at 4 °C. Internal binding was determined in the presence of MPPϩ (400 ␮M)

RESULTS

Predictions of distances at the hSERT Cys double mutants

Mutant pair

DISCUSSION