1) The study aimed to produce a water-soluble extracellular domain of nicotinic acetylcholine receptors (nAChRs) by inserting a flexible linker between the transmembrane and extracellular domains to allow protease cleavage. 2) Inserting a 6xAGS flexible linker into the α3 subunit significantly reduced expression of functional nAChRs containing the α3 subunit, as shown by low epibatidine binding. 3) Future work will test shorter flexible linker inserts, as the 6xAGS linker was likely too long and disrupted protein structure.

1. APPROACHES TO FORMING A WATER-SOLUBLE
EXTRACELLULAR DOMAIN FROM α3 SUBUNITS OF
NICOTINIC ACETYLCHOLINE RECEPTORS
Victoria L. Frankovich, Shama Dhanani, Alexandra M. Person and Gregg B. Wells
Department of Molecular and Cellular Medicine, Texas A&M Health Science Center and Texas A&M University
Background
Nicotinic acetylcholine receptors (nAChRs):
• Ligand gated ion channels in the nervous system and on neuromuscular junctions
• Members of superfamily of Cys-loop receptors which include GABA type A and C,
glycine and serotonin 5-HT3 receptors
• Composed of subunits designated α1 – α10, β1 – β4, δ, γ, and ε
• Neuronal nAChRs are composed of α and β subunits (1)
Nicotinic acetylcholine receptor subunit α3:
• Integral membrane protein containing
o 4 transmembrane segments
o A cytoplasmic loop between M3 and M4
o A large, ligand binding, extracellular, N-terminal domain (1)
• Found mostly in the peripheral autonomic nervous system
• Studying 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.
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. The nAChRs
are found on both the presynaptic and postsynaptic side of the synaptic space. (2)
Objectives
• Produce a water-soluble extracellular domain of a nAChRs in order to study the
structure by x-ray crystallography
• Determine a way to successfully insert a protease site at the M1-ECD junction.
• Could a flexible domain be a good environment for a protease site?
• 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, as a test of native structure, the ability of the mutated α3
subunit to bind to the ligand [3H]epibatidine and monoclonal anti-nicotinic
acetylcholine receptor antibody mAb35 (specific for α1, α3, and α5 subunits).
Fig. 2 Predicted flexibility of the α3 subunit containing a 6X-AGS linker
compared to the flexibility of the original α3 subunit
• Design primers containing a new 6X-AGS flexible linker to be added between the M1
domain and the extracellular domain
• Use PCR to insert the flexible linker into the α3 subunit coding sequence. Use plasmid
purification techniques and sequencing (Lone Star Labs) to confirm the presence of the
IRRL 6x-AGS insert.
• Linearize the plasmid DNA using AseI restriction digest and produce RNA from the
linearized DNA using the Ambion mMessage mMachine® SP6 Kit
• Inject the RNA into Xenopus laevis oocytes and extract, isolate, and purify the protein
produced in the oocytes
• Immunoblot with mAb35 to quantify expression
• Perform an epibatidine assay to quantify the yield of [3H]epibatidine binding sites
Methods
Results
Conclusions
New Questions
References
Acknowledgments
Binding Curves:
Fig. 3 Protein Expression of α3 and β2
nAChR subunits containing only the ECD
and M1 domain.
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 creating the
β2 6xAGS construct that was paired with my α3 construct. I would also like to thank
Kristen Cloyd for creating the α3 full length sequence in the pSP64A plasmid vector used in
this experiment.
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
New hypothesis: Length of an insertion at the extracellular domain/M1 interface affects
yield of a3-containing nAChR.
Future projects will evaluate the expression and binding affinity of two new α3/β2
constructs containing the following inserts at the ECD/M1 junction:
• A “short” insert only 5 amino acids in length
o A series of two restriction sites
• An insert consisting of only the TEV protease site
o The minimal length necessary for site-specific proteolysis to liberate the
extracellular domain
The shorter length of these inserts at the ECD/M1 junction will hopefully lead to
increased yield of [3H]epibatidine binding sites for a3-containing nAChRs.
0
5
10
15
20
25
30
35
40
45
50
Uninjected
Control
Control: a3
and B2
Subunits
a3 and B2
Subunits
with
Flexible
Linker
[³H]HEpibatidineBindingSites
PerOocyte(femtomoles)
Constructs Injected
0
10
20
30
40
50
60
70
80
90
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Uninjected
Control
Control:
a3 and B4
Subunits
Control:
a3 and B2
Subunits
a3 and B2
Subunits
with
Flexible
Linker
[³H]HEpibatidineBindingSites
PerOocyte(femtomoles)
Constructs Injected
[³H]Hepibatidine Binding Assay
First Injection
Future Plans
Why does the 6xAGS flexible linker cause the low expression level of mutated a3-
containing nAChR relative to wild-type a3-containing nAChR?
Was this flexible linker too long to be tolerated by the protein?
The low yield of epibatidine binding sites demonstrated by the α3 and β2 constructs
containing the 6xAGS flexible linker at the ECD/M1 junction suggests that the flexible
linker would be a poor environment in which to place a protease site.
Epibatidine Binding Assay:
[³H]Hepibatidine Binding Assay
Second Injection
Fig. 5 Epibatidine binding assay. Very low yield of [3H]epibatidine binding sites was
seen when the α3full 6xAGS was paired with the β2full 3xFLAG 6xAGS subunit as
compared to the control subunits without the flexible linker.
Fig. 4 [3H]epibatidine Binding curves of
full length and ECD α3β4 nAChR and M1
and ECD α3β4 nAChR. Data supports the
presence of two sites in full length and
ECD α3β4 nAChR.
a3M1
b2M1
+
b2M1
49
35
29
PNGase F
Expected
30 kD
a3M1
Expected
30 kD
50
37
25
20
75
uninjected
control