1. The activating Zn 2+ switch by mutation of tyrosine to alanine possibly causing aversion to cocaine paired salt ions by C.elegans.

2. Does the mutation of Tyrosine to Alanine in Dopamine transporter impair cocaine’s affinity to bind to the Dopamine Transporter?

4. Cocaine use has multiple side effects including: Increased blood pressure, Increased heart rate Risk of cardiac arrest

5. Model organism C.elegans Non-parasitic transparent nematode(worm) It is 1mm in length Lives in temperate soil environment It has its whole genome sequenced It also has its neural wiring diagram completed

6. Why the use of Zinc? Research has shown that binding of Zn 2+ to the endogenous Zn 2+ binding site of the human dopamine transporter causes an inhibition of dopamine uptake. The mutation of Tyrosine to Alanine, in an experiment done by Loland et a,l switches the Zn 2+ form an inhibitor of dopamine uptake to an activator.

7. Loland et al they mutated the human DAT’s tyrosine to alanine at position 355 and transfect the mutated hDAT into COS7 cells (cell lines derived form monkey kidney tissue )to see if cocaine would have a binding affinity to DAT in the presence and absence of Zn 2+

8. CRISPER/Cas9 Clustered regularly interspaced short palindromic repeats CRISOR/Cas9 originally used by bacteria

9. Lipofection Method Lipofectamine is a transfection reagent used for both DNA and RNA transfection. Magnetic Nanoparticles

10. Figure adapted form Loland et al Measure Cocaine’s ability to inhibit dopamine uptake.

11. If all goes well, I expect to get similar results with my transfected cells of C.elegans where the cocaine’s ability to inhibit Dopamine uptake is impaired.

12. Limitation The experiment might not go as planned mainly because its dependent on Zn 2+ being able to the Zinc binding site on C.elegans DAT like it did on the Human DAT in Loland et al.

13. References Reference Loland, C. J., Norregaard, L., Litman, T., & Gether, U. (2002). Generation of an activating Zn2+ switch in the dopamine transporter: Mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1683– 1688. doi:10.1073 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122251/pdf/pq0302001683.pdf Musselman, H. N., Neal-Beliveau, B., Nass, R., & Engleman, E. (2012). Chemosensory Cue Conditioning with Stimulants in a Caenorhabditis elegans Animal Model of Addiction. Behavioral Neuroscience, 126(3), 445–456. doi:10.1037 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367381/pdf/nihms368977.pdf Nian-Hang Chen, Maarten E. A. Reith, Michael W. Quick. (2004). Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch - Eur J Physiol 447:519–531 DOI 10.1007/Nian-Hang ChenMaarten E. A. ReithMichael W. Quick http://link.springer.com/article/10.1007%2Fs00424-003-1064-5#

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