A summary of my undergraduate research conducted in the Wells Lab my junior year presented at the 2013 Biochemistry and Genetics Society Poster Session.

1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion
channels which provide an important ion transport function in both the
nervous system and on neuromuscular junctions. They are members of the
superfamily of nicotinoid receptors which include GABA type A and C,
glycine and serotonin 5-HT3 receptors. This receptor is composed of
subunits which are designated α2 – α10, β2 – β4, δ, γ, and ε. The neuronal
nAChRs are composed of α and β subunits (1).
Nicotinic Acetylcholine Receptor subunit α3 is an integral
membrane protein containing 4 transmembrane segments, a cytoplasmic
loop between M3 and M4 and a large, ligand binding, extracellular, N-
terminal domain (1). The α3 subunits are found mostly in the peripheral
autonomic nervous system. Studying the structure of the α3 subunit will
provide knowledge needed for understanding how these receptors function
and how they can be the target of drugs intended for specific effects within
the nervous system.
Introduction
Objectives
Primers were designed that contained the genes for encoding the new 6X-
AGS flexible linker and also the IRRL residues which occur naturally right
before the M1 domain. The primers were designed to insert the linker
between the M1 domain and the extracellular domain. They were then
produced by Invitrogen.
PCR was used to successfully insert the IRRL 6X-AGS sequence into the
α3 subunit coding sequence. The DNA was amplified by bacterial
transformation in One Shot® OmniMAX™ 2 T1 Chemically Competent
E.coli and then purified and isolated with QIAfilter® Plasmid Midi Prep
Kit from Qiagen. Sequencing was performed by Lone Star Labs to
confirm the presence of the IRRL 6x-AGS insert.
The plasmid DNA containing the α3 Full and IRRL 6X-AGS sequence was
linearized using AseI restriction digest from New England Biolabs and the
correct linear DNA production was confirmed by gel electrophoresis.
RNA production from the linearized DNA was performed using the
Ambion mMessage Machine® SP6 Kit . The RNA product was visualized
and analyzed by gel electrophoresis.
The RNA will be injected into Xenopus laevis oocytes. The protein
produced in the oocytes will be extracted, isolated, and purified. Western
blotting with mAB35 will be used to quantify expression. Finally a
Epibatidine Assay will be completed to quantify binding affinity.
Materials & Methods
The 6xAGS linker used in my project was also inserted into the α4 subunit
using the same procedure in a project completed by Shama Dhanani in
2011 under the direction of Dr. Wells. A western blot and epibatidine
binding assay were completed on this protein construct.
Conclusions and Predictions
Future Plans
After the discovery of the double bands in the α3full IRRL 6xAGS RNA
the α3full IRRL 6xAGS pSP64A plasmid was relinearized using brand
new AseI restriction digest. The correct bands were also observed in this
digest. Next, RNA will be created with the relinearized DNA.
Hopefully the double bands will not occur again when I create the RNA
from my relinearized construct. But even if the double bands do occur
again, we will proceed with the oocyte injection and protein collection.
We also plan to go back to the α3full kozak XenOpt pSP64A plasmid that
my construct originated from and linearize and create RNA from that
DNA. This way we can check if there were any discrepancies in the
original construct.
References
1. Wells, Gregg B. "Extracellular Domain Nicotinic Acetylcholine
Receptors Formed by α4 and β2 Subunits." Journal of Biological
Chemistry 280.48 (2005): 39990-40002
2. Laviolette, Steven R., and Derek Van Der Kooy. "The Neurobiology of
Nicotine Addiction: Bridging the Gap from Molecules to Behaviour."
Nature Reviews Neuroscience 5.1 (2004): 55-65.
3. Ishida, T and Kinoshita, K, PrDOS: prediction of disordered protein
regions from amino acid sequence., Nucleic Acids Res, 35, Web Server
issue, 2007
Acknowledgments
Fig. 7 Epibatidine binding assay. Very low binding affinity was shown
when the α4full 3xFLAG 6xAGS was paired with the β2full 3xHA subunit
and even worse binding affinity when paired with the β2full 3xFLAG
6xAGS subunit.
To study the structure of the extracellular domain of nAChRs by x-ray
crystallography, there needs to be a way to produce just the extracellular
domain. A protease site was inserted just outside of the M1 domain of a
M1 and extracellular domain construct, but it reduced expression of the
protein. Putting the protease site within a more flexible environment could
increase protein expression. The ability of the protein to tolerate this
flexible insert must first be determined.
The objective of this experiment is to insert a flexible 6xAGS link into
the full length nAChR α3 subunit protein in between the M1 domain and
the extracellular domain and evaluate the ability of the mutated α3 subunit
to bind to Monoclonal Anti-Nicotinic Acetylcholine Receptor α1, α3, α5
subunits (clone mAB35). Figure 2 shows the predicted flexibility of the
mutated α3 subunit sequence (3).
Fig. 5 Western Blot used to test the presence of the Beta subunit. The mAB
anti-HA antibody was used to bind the HA epitope tag within the beta
subunit. A strong presence of the subunit was detected and can be seen
around 60 kDa.
Western Blot with antibody: mAb Anti-HA
Fig. 4 Gel Electrophoresis of RNA products. α4 M1 2x142 Linear DNA was
used to produce RNA alongside the α3 Full IRRL 6x-AGS Linear DNA. The
RNA was also compared to a previously created control α4 M1 2x142
RNA. There was successful RNA production of the α4 M1 control but the
α3 Full IRRL 6X-AGS RNA shows unexpected double bands.
RNA Production:
Fig. 2 Predicted flexibility of the α3 subunit containing a 6X-AGS linker
compared to the flexibility of the original α3 subunit .
Fig. 3 Gel Electrophoresis of the PCR Products. Bands at 4605 bp, the
length of the goal construct, can be observed in PCR products #2-4. PCR
product #3 was used for the rest of the experiment.
PCR Products:
Fig. 1 a) Each nAChR is composed of α and β subunits. Each subunit
contains 4 transmembrane domains and a large ligand binding
extracellular domain. b) The nAChRs are found on both the presynaptic
and postsynaptic side of the synaptic space. (2)
4000
Related Data
Results
Department of Molecular and Cellular Medicine, Texas A&M Health Science Center and Texas A&M University
Victoria L. Frankovich, Shama Dhanani, Kristen Cloyd, Alexandra M. Person and Gregg B. Wells
Effects of modifying backbone flexibility in α3 subunits
of nicotinic acetylcholine receptors
1
10
100
1000
10000
Control: a4M1-2x
142mAb + B2M1-
236mAb
Project Controls:
a4 full 3xFLAG
pSP64A + B2full
3xHA pSP64A
a4 control with
new B2: a4 full
3xFLAG pSP64A +
B2full 3xHA
6xAGS IIRRK
B2 control with
new a4: a4full
3xFLAG 6xAGS
IRRL + B2full
3xHA pSP64A
New a4 and B2:
a4full 3xFLAG
6xAGS IRRL +
B2full 3xHA
6xAGS IIRRK
DPM1
nAChR Subunits Used
Epibatidine Binding Assay
All measurements were taken with
1nM Epibatidine, and with 299 mAb
I would like to thank Dr. Gregg B. Wells and Alexi Person for their
contributions and support in carrying out this project. I would like to thank
Shama Dhanani for letting me use the data from her α4 project as a
comparison to my project. I would also like to thank Kristen Cloyd for
creating the α3 full length sequence in the pSP64A plasmid vector used in
this experiment.
So far in this experiment I can conclude that the AGS linker was
successfully inserted into the α3 subunit gene. However, because of the
strange double bands seen in the gel of the RNA I cannot conclude that the
RNA being produced is the correct α3 RNA product.
After reviewing the data collected from the a4full 3xFLAG 6xAGS project
and its low binding affinity during the epibatidine binding assay, it can be
predicted that the 6xAGS flexible linker in the a3full IRRL 6xAGS
construct will also not allow successfully ligand binding.
However, there is hope that inserting the protease site into the full length
protein will not reduce expression as much as inserting the protease site
into the M1 and extracellular domain construct.
600
Fig. 6 Western Blot used to test the presence of the alpha subunit. The mAb
anti-FLAG antibody was used to bind to the FLAG epitope tag located in
the α4 subunit. A strong presence of the subunit was detected and can be
seen at the 75 kDa mark.
Western Blot with antibody: mAb anti-FLAG