Pieces from my Poster

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:

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. 

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 :)

Catch up of July

I can't believe I am already on week 9. This blog will be extra long, because I have to fill you in on July 12-August 3.
Outside of the lab I have:

• Traveled to Fargo
• Ridden a ferris wheel inside the original Sheels sporting goods store.
• Been kidnapped, blind folded, and taken to the school planetarium to watch HP 7.1 on the big screen
• Watched HP 7.2
• Eaten at the locally famous "Red Pepper", Blue Moose, Little Bangkok, Rhombus Guys. As well as Happy Joe's and Applebees.
• Had a hall dance party with glow sticks, strobe light, and black light
• Built a fort
• Watched the sun rise from the top of the parking garage
• Played kickball
• Went swimming at a hotel pool
• Went star gazing
• Walked across the Mississippi river headwaters
• Bowling
• Sardines in Walmart
• Slack line
• 9 mile tandem bike ride at 11pm
• Baked a cake
• Cooked pancakes and bacon on my griddle

In lab:

• PCR: Creating the mutation on a template DNA plasmid
• Transformations: inserting the DNA into E. coli
• Culturing E. coli: Growing cells on petri dishes, picking colonies, and growing those colonies in flasks
• Minipreps out the wazoo: purifying DNA from E coli
• Restriction digests: cutting the DNA with special enzymes that recognize specific base pair sequences
• Gel electrophoresis: pushing the digested DNA through agarose to separate cut DNA pieces of different lengths (This is a check to make sure we have the right DNA) We can view the bands of DNA under a UV light.

• Sequencing the DNA: a long complicated processes that I don't really understand, but it gives me the base pair sequence of the DNA to make sure the correct mutation was created and no additional ones were made. Each wave in the chromatogram pictured below represents a different base pair.

• Culturing HeLa cells: Taking care of the immortal cell line of HeLa cells. Passing them into new flasks every 3 days to keep them from getting over crowded, and giving them new food to eat. 

• Plating HeLa cells: Counting my cells and putting 50,000 into each well of a 24 well plate.
• Transfecting my mutated DNA into my HeLa cells: Inserting the mutated DNA into the HeLa cells on the plates.
• Uptake of radioactive serotonin with a serial dilution of inhibitor: The DNA that was inserted into the HeLa cells sits over night and the cells will read the DNA and start creating serotonin transporters in their cell membrane. I then add inhibitor (citalopram-an antidepressant drug generically referred to as Lexapro) at various concentrations. Then I add radio active serotonin, which should be transported into the cell through the newly created transporters. 
• Counting radioactivity inside the cells: The 24 well plates are then put in a machine called a Top Counter, which counts the amounts of radioactivity inside the cell, which tells us how well the transporter worked or didn't work. 
• Creating a competition curve from the Top Counter data: A graph of the results will show a curved line on the axies of inhibitor concentration and radioactive serotonin uptake. The curve will shift left or right depending of if the mutation in the transporter is helping or hindering the uptake. If it is being hindered, then we can assume that the place where the mutation was created is most likely a binding site of the inhibitor.

It has all been really exciting to watch my project get to the results stage. I won't have time to run the experiment on all of my created mutations, but someone else in the lab will continue my project after I leave.

I am to the point where I feel accomplished, and I can leave feeling successful. I have learned sooo many lab techniques and computer programs. I have gotten to know my lab mates, and fellow REU students really well, and I am going to miss all of them.