Abstract:
As the target of many mood effecting drugs such as antidepressants, cocaine, and 3,4 methylenedioxymethamphetamine (MDMA, ‘ecstasy’), the human serotonin transporter (hSERT) is of significant clinical importance and the subject of many pharmacological studies. Current understanding of how antagonists bind to hSERT is advancing rapidly due to recent crystal structures but remains unclear. Data suggest binding of antagonists occur at a binding site located at the center of the transporter. In fact, our lab as well as other labs have elucidated several residues at this site that are critical for high-affinity citalopram (CIT) binding1,2. However, there are studies suggesting the binding site is located more cytosolic and positioned above the outer gate3. Recently, to further characterize CIT binding, we performed quantitative structure activity relationship studies (QSAR) using CIT analogs and the hSERT mutants Y95F, I172M, S438T. Results from this study suggest that the I172M and S438T mutations may result in complete loss of CIT binding at central high-affinity binding site (HABS) causing CIT to bind at a secondary site. In contrast, CIT binding appears to be maintained at the HABS in the Y95F mutant. In order to test the presence of a secondary binding site, we generated cytosolically-located mutations in wild type, Y95F and I172M hSERT backgrounds and evaluated their impact on CIT binding using [3H]5-HT competition uptake assays.
Introduction:
Previous research on hSERT has identified Y95 on transmembrane 1a (TM1a) and I172 on TM 3, as critical for high-affinity binding of many antidepressants. It was found that an amino acid mutation of Tyr to Phe at residue 95, or Ile to Met at residue 172, resulted in a significant reduction of antidepressant potency1. Our studies on a library of CIT analogs revealed the compounds interacted with the I172M and S438T mutants in a manner distinct from WT and Y95F where many CIT analogs exhibited lower inhibitory potency (IC50) in WT and Y95F backgrounds. In contrast, the IC50 of compounds in the I172M/S438T backgrounds was largely unaffected and in some cases displayed increased potency. This led to the hypothesis that mutation at either I172M or S438T eliminate the ability of CIT to bind at the HABS and divert CIT binding to a secondary binding site elsewhere in hSERT. CIT in the WT or Y95F mutant is thought to bind in the HABS. This hypothesis explains the QSAR analysis that suggest alteration of pharmacophores on CIT differentially impact WT/Y95F versus I172M/S438T mutants.
hSERT is in the same transporter family as the bacterial Leucine transporter (LeuT) from Aquifex aeolicus. LeuT protein was stable enough to form crystals and obtain high resolution x-ray diffraction structure and some subsequent co-crystal structures revealed determined that tricyclic antidepressant inhibitors bind in an upper region of the transporter on the extracellular face of the transporter3. However, the majority of biochemical data suggests that SERT antagonists bind at a more central binding site. The possibility that two sites exist suggests that our hypothesis of a secondary binding site existing in the upper region of hSERT is reasonable.
Methods:
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| Side view of transporter |
Selection of residues for mutation to identify LABS: An hSERT comparative model (LeuT) was visualized in MacPyMOL and candidate residues (magenta side chains) were chosen based on their proximity to the cavity above the outer gate residues R104 and E493 (orange and blue side chains). Amino acid substitutions were introduced to alter residue hydrophobicity, size, and charge. Mutagenic forward and reverse primers were designed for each substitution. Silent restriction sites were also built into the primers to identify whether or not the desired mutation was generated.
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| Top view of transporter |
Site-directed mutagenesis and construction of mutant plasmids: Mutants were created using PCR mediated site-directed mutagenesis QuikChange II (Stratagene). DNA purification and analysis: Plasmids were amplified in bacteria and isolated using the Wizard+ SV Miniprep kit (Promega). DNA concentration and purity were determined spectrophotometrically. DNA was digested with the appropriate silent site enzymes and separated on 0.75% agarose gel. Bands were visualized under UV. Positive clones were verified by sequencing at NorthWoods DNA Inc.
Expression of DNA in HeLa cells: Mutant SERTs were transiently expressed in HeLa cells and analyzed for uptake of radiolabeled serotonin. Paroxetine and non-transfected cells were used to determine specific uptake. Functional mutants were tested in a [3H]5-HT competition uptake assay to determine changes in CIT potency.
Results: All of my results and discussion is hard to explain without the graphs and poster, so you will just have to talk to me about it in person if you are interested :)
