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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