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Brown Deer High School SMART Team: Georgina Foran, Noah Freuler, Teylor Harris, Ashley Higgins, Justin Johnson,
Isaac Ngui, Brett Poniewaz, Gloria Ramos, Luke Richmond, Noel Stoehr, Virginia Tuncel
Teacher: Dave Sampe
Mentors: Andy Weyer, Ph.D. and Katherine Zappia, Ph.D. Candidate, Medical College of Wisconsin, Department of Cell Biology, Neurobiology and Anatomy
Pain, a problem faced by most humans, can be felt
in two different ways- acute pain, which is the sharp
pain usually brought about by injury, and chronic
pain, which is pain that persists for three or more
months. Chronic neuropathic pain affects 3-4% of
people worldwide2. Treating pain that lasts this long is costly.
Annually, treatment for chronic pain costs roughly $560$635 billion in the U.S. alone5. This is partly due to the
ineffective treatments that are currently available. Opioids,
used effectively to treat acute pain, are highly addictive and
lose efficacy when used as a treatment for chronic
pain. Pregabalin is a promising non-opioid drug
option, but one study showed it only works in 26.1%
of patients7. Researching pain is difficult because of
differences in people’s genetics and pain thresholds.
Perception of Pain
Pain is sensed by sensory neurons in our skin and other tissues
(see figure 3). These neurons can be activated by mechanical,
chemical or thermal stimuli. The neurons have mitochondria
specific receptors designed to pick up on the
vesicle
stimuli (see figure 1). The stimuli open the ion
channels and allow positive ions in, which cause
depolarizations that make the neuron’s interior
less negative. When enough stimuli are received
and the threshold of the neuron is reached,
receptors
that neuron produces an
action potential of its own Figure 1. Synapse
(see figure 2). An action potential is a
very quick influx of sodium ions and
efflux of potassium ions through voltage
gated channels. This generates an
electric current in a wave down the
axon. This impulse travels to the spinal
Figure 2. Graph of action potential
cord and when it reaches the end of the
neuron, a chemical neurotransmitter is released into the
synaptic cleft, stimulating the next neuron.
Somatosensory
Cortex
This second neuron sends the impulse
to the brain so the pain sensation
can be interpreted.
1
10
The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is one of the many receptors
that allow us to feel pain (see figure 7). TRPA1 can be triggered either mechanically, chemically or thermally.
TRPA1 is an ion channel located in the free nerve endings in your skin. Several agonists that chemically
trigger TRPA1 include: mustard oil, tear gas, hydrogen peroxide, nicotine, and wasabi (see figure
4). When the skin is stretched, the ion channel is opened (see figure 5).
Researchers speculate that the ankyrin repeats are the mechanism that
allow the channel to mechanically open. Positive amino acid residues in
the coiled coil may assist ion passage by attracting polyphosphates (see
Figure 4. Wasabi
figure 6) which help to open the channel. Some amino acids make the
channel so narrow that most ions are restricted from passing through. Ca++ and Na+ are
able to pass through the channel depolarizing the neuron, which may result in an
Figure 5. Top view of pore channel
impulse sent to our brain. Researchers know that TRPA1 is involved in mechanical pain
in the middle of 4 subunits
(PDB File 3J9P)
because when they remove the genes for TRPA1 in mice, they are less sensitive to pain.
These “knock out” animals can be poked on a sensitive paw and they don’t elicit a withdrawal response. (See
“Evidence that TRPA1 works” for more detail.) TRPA1 is also responsible for pain sensation in
humans. A study was performed on a family who had a N855S mutation in the S4 region of
TRPA1. This mutation is an autosomal dominant disease carried on chromosome 8. With
normal stimuli, the mutant channel created five times as many depolarizations which caused
pain. In these patients, debilitating upper body pain is brought on by fasting, illness, cold, or
Figure 6. Inositol
fatigue in this example of pain sensed by TRPA1 receptors.
hexaphosphate
12
To show how TRPA1 facilitates
pain sensation, an experiment
was performed on mice with a
control group and a group with
the TRPA1 gene knocked out
(see figure 8). Without a
functioning TRPA1 gene, knock
out mice respond to pain less
often.
Figure 8. Mice respond to pain less often
when the TRPA1 gene is knocked out.
In another experiment, the
paws of mice were
sensitized with Complete
Freund’s Adjuvant (CFA), a
compound that induces
inflammation (see figure 9).
When the paw is touched,
fewer actions potentials are
sent in mice lacking TRPA1
receptors. Therefore, mice
responded less often (see
figure 10).
Figure 9. Skin nerve preparation
(a polyphosphate)4
Pore mouth- negative
residues attract cations
(Ca++ and Na+) to pore.
The Structure of TRPA1
Upper and Lower
Restriction- Asp915,
Ile957 and Val961 form
the narrowest portion of
the channel, only
allowing Ca++ and Na+
ions to pass through.
Antagonist Binding SitePhe909 is where a
specific antagonist, A96709, binds to block
pain.
Transmembrane regionalpha helices in this
region span the neuron’s
membrane.
Mutation SiteMutations that turn
Asn855 to serine can
cause familial episodic
pain syndrome (FEPS).
Amino Acids in the
Coiled Coil- TRPA1
activity may be
modulated when
polyphosphates bind to
Lys1046, Arg1050,
Lys1048 and Lys1052,
acting as second
messengers.
Ankyrin Repeat Domainmay be involved in
mechanical stimulation,
acting like a spring or
bending toward pore
when TRPA1 is
mechanically activated.
Figure 3. Brain- coronal section3, Skin- cross section13, Spinal cord- transverse section11
Involvement of TRPA1 in Pain
The Story of TRPA1
Pain… We’ve All Felt It
Agonist Binding SiteCys621, Cys641 and
Lys710 are where
agonists bind causing
pain sensation.
Alpha helices are
highlighted in yellow.
Figure 7. TRPA1 model made by the Brown Deer High School SMART Team
(Students Modeling A Research Topic) modified from Protein Data Bank file 3J9P
and 3D printed at Milwaukee School of Engineering
The SMART Team Program is supported by the National
Center for Advancing Translational Sciences, National
Institutes of Health, through Grant Number
8UL1TR000055. Its contents are solely the responsibility
of the authors and do not necessarily represent the
official views of the NIH.
Figure 10. Fewer action potentials are sent along the
sensory neurons when TRPA1 receptors are blocked
with an antagonist9.
Summary: Why Study TRPA1?
By studying TRPA1, researchers hope to find more effective
pharmaceuticals or alternative treatments to alleviate the pain
in individuals suffering from chronic pain. Research hopes to
decrease the health care costs of pain treatment, as well as the
lost productivity of people out of the workforce. By mitigating
chronic pain, people will be able to live a quality pain-free life.
If restored to health, people will have the ability to work, enjoy
recreational activities and everyday life without pain.
References
1. Axon Terminal. (2016, February 3). Retrieved February 4, 2016, from https://en.wikipedia.org/wiki/Axon_terminal
2. Global Industry Analysts, Inc. (2011). Global Pain Management Market to Reach US$60 Billion by 2015, According to a
New Report by Global Industry Analysts, Inc. Retrieved from http://www.prweb.com/pdfdownload/8052240.pdf
3. Hewitt, J. (2013, October 17). Mapping Einstein’s brain | ExtremeTech. Retrieved February 4, 2016, from
http://www.extremetech.com/extreme/168046-mapping-einsteins-brain
4. Inositol hexaphosphate. (2015). Retrieved February 4, 2016, from http://www.chemspider.com/ChemicalStructure.18505485.html
5. Institute of Medicine. Relieving Pain in America: A Blueprint for Transforming Prevention, Care, Education, and Research.
Washington, DC: The National Academies Press, 2011. doi:10.17226/13172
6. Lennertz, R.C., Kossyreva, E.A., Smith, A.K., Stucky, C.L. (2012). TRPA1 mediates mechanical sensitization in nociceptors
during inflammation. PLoS ONE 7(8): e43597. doi: 10.1371/journal.pone.0043597.
7. Moon, D. E., Lee, D. I., Lee, S. C., Song, S. O., Yoon, D. M., Yoon, M. H., . . . Lee, P. B. (2010). Efficacy and Tolerability of
Pregabalin Using a Flexible, Optimized Dose Schedule in Korean Patients With Peripheral Neuropathic Pain: A 10-Week,
Randomized, Double-Blind, Placebo-Controlled, Multicenter Study. Clinical Therapeutics, 32(14), 2370-2385.
8. Paulson, C.E., Armache, J., Gao, Y., Cheng, Y., Julius, D. (2015). Structure of the TRPA1 ion channel suggests regulatory
mechanisms. Nature 520: 511-513.
9. PDB ID: 3J9P
10. Samuel, L. (2011, November 11). 013 A Review of the Action Potential - Interactive Biology, with Leslie Samuel. Retrieved
February 4, 2016, from http://www.interactive-biology.com/1597/a-review-of-the-action-potential-episode-13/
11. Walker, C. (2016). Peripheral Nervous System. Retrieved February 4, 2016, from http://www.biologyreference.com/OcPh/Peripheral-Nervous-System.html
12. What is Wasabi? (2016). Retrieved February 3, 2016, from http://www.wisegeek.org/what-is-wasabi.htm
13. Winslow, T. (2008). Retrieved February 4, 2016, from
http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0022671/?figure=1