The activating Zn2+ switch by mutation of tyrosine to alanine possibly causing

aversion to cocaine paired salt ions by C.elegans .

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

• Cocaine use has multiple side effects including:• Increased blood

pressure,• Increased heart

rate • Risk of cardiac

arrest

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

Why the use of Zinc?Research has shown that binding of Zn2+ to the

endogenous Zn2+ 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 Zn2+ form an inhibitor of dopamine uptake to an activator.

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 Zn2+

CRISPER/Cas9Clustered regularly interspaced short palindromic

repeats

CRISOR/Cas9 originally used by bacteria

Lipofection MethodLipofectamine is a transfection reagent used for

both DNA and RNA transfection.

Magnetic Nanoparticles

Figure adapted form Loland et al

Measure Cocaine’s ability to inhibit dopamine uptake.

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.

Limitation The experiment might not go as planned mainly

because its dependent on Zn2+ being able to the Zinc binding site on C.elegans DAT like it did on the Human DAT in Loland et al.

References Reference

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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/

http://link.springer.com/article/10.1007%2Fs00424-003-1064-5#

 

 

 

 

 

Gether, Ulrik, Andersen, Peter H., Larsson, Orla M, Schousboe, Arne. (2006). Neurotransmitter transporters: molecular function of importantdrug targets. TRENDS in Pharmacological Sciences Vol.27 No.7. 375-383

http://www.sciencedirect.com/science/article/pii/S0165614706001271

Norregaard, L., Frederiksen, D., Nielsen, E. O. & Gether, U. (1998) EMBO J. 17, 4266–4273.

Lo, T.-W., Pickle, C. S., Lin, S., Ralston, E. J., Gurling, M., Schartner, C. M., … Meyer, B. J. (2013). Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions. Genetics, 195(2), 331–348. doi:10.1534

http://www-ncbi-nlm-nih-gov.proxy.library.vcu.edu/pmc/articles/PMC3781963/

Chiu, H., Schwartz, H. T., Antoshechkin, I., & Sternberg, P. W. (2013). Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas. Genetics, 195(3), 1167–1171. doi:10.1534

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813845/pdf/1167.pdf

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