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The Activating switch of
Zn2+ by the mutation of the
amino acid Tyrosine to
Alanine in the Dopamine
Transporter protein
possibly impairing
cocaine’s effect on
locomotion
Cocaine in the Nervous
System
 Stimulant
Science.education.nih.gov
 Short term use
 Increased blood pressure
 Increased heart rate
 Risk of cardiac arrest
 Long term use
 Changes the chemistry of the brain
What’s the significance of Zinc in
Dopamine Transporter ?
Loland et al
Biology.syr.edu
Can this be done in C.elegans to
further examine if the switch of
Zn2+ by the mutation of Tyrosine to
alanine can possibly cocaine’s
effect in locomotion ?
 They are the simplest organisms with a nervous system
 They have their whole genome sequenced.
 They are easily accessible.
 Has short generation time.
 Cost and time effective.
Which gene codes for DAT in
C.elegans?
 Dat-1 gene codes for
C.elegans
 Located on
Chromosome III
Ncbi.nlm.nih.gov
Does the Same Tyrosine used in
hDAT mutation exist in
C.elegans?
C.Elegans DAT
Human DAT
EMBOSSEbi.ac.uk
What mRNA codons are
responsible for the production of
Tyrosine?
Start
ORF
program.
(Star.mit.eu)
 Possible
codons for
Tyrosine:
 UAC
 UAU
Biochem.com
 UAC
 UAU
Tyrosine
GC
U
GC
C
GC
A
GC
Alanine
CRISPR/CAS9Method
Sander et al
Sander et al
Measure effectiveness of mutation
by analyzing cocaine’s ability to
inhibit dopamine uptake in Y328A
DAT both in the presence and
absence of Zn2+
Transfection Method
Niborlab.com
Ibioexperts.com
Possible Outcomes
Dopamine Uptake
percent is going to be
greater in the mutated
DAT compared to wild
type as concentration
of cocaine increases.
Dopamine Uptake
percent of the
mutated DAT is
going to be
identical to wild
type as
concentration of
cocaine increases.
Dopamine
Uptake percent
is less in the
mutated DAT
compared to wild
type as
concentration of
cocaine
increases.
Cocaine’s ability to inhibit DA
uptake has been impaired
Loland et al
Cocaine’s ability to impair
DA uptake has not been
affected
Loland et al
Cocaine’s ability to impair
DA uptake has been
amplified.
Loland et al
Possible Outcomes
Dopamine Uptake
percent is going to be
greater in the mutated
DAT compared to wild
type as concentration
of cocaine increases.
Dopamine Uptake
percent of the
mutated DAT is
going to be
identical to wild
type as
concentration of
cocaine increases.
Further experiments to see
whether or not mutated
dopamine transport makes
C.elegens less susceptible to
behavioral effects of cocaine
(i.e locomotion)
Dopamine
Uptake percent
is less in the
mutated DAT
compared to wild
type as
concentration of
cocaine
increases.
References
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 chloridedependent 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 important
drug 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