the activating zn 2+ switch by mutation of tyrosine to alanine possibly causing aversion to cocaine...
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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
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http://link.springer.com/article/10.1007%2Fs00424-003-1064-5#
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http://www.sciencedirect.com/science/article/pii/S0165614706001271
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http://www-ncbi-nlm-nih-gov.proxy.library.vcu.edu/pmc/articles/PMC3781963/
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