ACTIVATING TRPA1 CHANNELS DROSOPHILA PAINLESS MUTANTS: A MODEL FOR SCREENING CHEMESTHETIC IRRITANTS ACTIVATING TRPA1 CHANNELS Lauren Leifeste, Dr. Wayne Silver Department of Biology, Wake Forest University, Winston-Salem, NC 27109 leifle6@wfu.edu Introduction Chemesthesis is the sense of irritation produced by chemicals. In mammals, the trigeminal nerve is a mediator for chemesthesis in the head and face.  Chemical irritants stimulate the trigeminal nerve, although the mechanism of stimulation for many irritants is unknown. The transient receptor potential (TRP) family constitutes a large variety of polymodal receptors which respond to various noxious stimuli including irritating chemicals. The TRPA1 channel found on mammalian trigeminal nerves responds to numerous irritants, e.g. allyl isothiocyanate (AITC), the active ingredient in mustard oil and wasabi (Karashima et al, 2007).  The fruit fly, Drosophila, expresses at least three TRPA1 channels; dTRPA1, Pyrexia, and Painless (Sokabe et al., 2008). The painless gene expressed in gustatory receptor neurons is an evolutionary homolog of mammalian TRPA1 and plays a role in the flies’ ability to avoid AITC (Al-Anzi et al, 2006). We are using a Drosophila feeding assay to examine what other known trigeminal irritants might also stimulate these TRPA1 channels. Previously, we demonstrated significant differences in the ability of Painless mutants (lacking the painless gene) and wild-type flies to avoid 11 chemical irritants using a feeding assay. In the present experiment we confirmed this finding in rescued flies which had painless re-engineered into their genome. Theoretically, responses of the rescued flies should be identical to responses of the wild-type flies tested previously 1% sucrose 1% sucrose + irritant Combination of blue and red solutions Figure 1. 50 Flies were starved in 2% agar vials for 24 hours. They were then used in one hour feeding preferences tests in complete darkness. After completion of the test the abdomens of the flies were scored for feeding preference based on their color. (from Al-Anzi et al., 2006) Materials & Methods The feeding assay is described by Al-Anzi et al. (2006) ( Figure 1). Two groups of 50 D. melanogaster were placed into separate vials containing a 2% agar solution. The flies were then starved in these vials for a 24 hour period in an incubator. After the 24 hour starvation period, 50 flies were placed on one of two 96-well plates with lids that served as the test arena (see fig. 1). For one plate each well contained either 2% agar + 1% sucrose + red dye or 2% agar +1% sucrose + blue dye + 2 mM of a chemical irritant. The other well plate contained the same compounds except the placement of the dyes was reversed. We tested 11 known trigeminal nerve irritants. The flies were left in the feeding assay for one hour in complete darkness in an incubator. After an hour, the flies were anesthetized and scored for feeding preference based on the color of their abdomens. Each red and blue abdomen was counted as 1 fly for its respective stimulus; purple abdomens were counted as ½ of a fly for each stimulus. Flies with clear abdomens were counted as 0. A Preference Index was calculated as the fraction of flies that had eaten the test stimulus, minus the fraction of flies that had eaten the control sucrose solution. That is: A PI close to +1 indicates that the flies were attracted to the test material while -1 indicates an avoidance. Conclusions There were significant differences in the ability of Painless and wild-type, and subsequently the rescued, flies to avoid most but not all of chemical irritants tested. Thus, the feeding assay designed by Al-Anzi et al. (2006), appears to be a useful tool for testing for the avoidance of chemical irritants. For most of the chemicals tested, the rescued flies had a more severe avoidance response. The response to nicotine in the rescued flies proved to be more like the painless than the wild type. Al-Anzi et al. (2006) and the current study suggest that Painless is involved in detecting chemical irritants. However, Sokabe et al.(2008) reported that Painless expressed in HEK cells is not activated by irritants such as AITC. Perhaps Painless is not a direct receptor for these compounds and interaction with accessory proteins, the formation of heteromeric channels and/or splice variants may be responsible for the in vivo response. Our results indicate that rescued flies behaved like wild-type flies for the majority of the chemical irritants demonstrating that Painless is responsible for the detection of these irritants. The Painless mutant appears to be a good model for determining which irritants activate TRPA1 in D. melanogaster * * * Not yet tested Figure 2. The average preference index for wild type, Painless, and rescued flies for the 11 chemicals tested. A negative preference index indicates avoidance, with the maximum avoidance possible being -1. The error bars indicate standard error. Literature Cited Al-Anzi B, Tracey DW Jr., Benzer S. (2006) Repsonse of Drosophila to Wasabi Is Mediated by painless, the Fly Homolog of Mammalian TRPA1/ANKTM1. Current Biology. 16:1034-1040. Karashima Y, Damann N, Prenen J, Talavera K, Segal A, Voets T, Nilius B. (2007) Bimodal action of menthol on the transient receptor protential channel TRPA1. Neuroscience. 27:9874-84. Sokabe T, Tsujiuchi S, Kadowaki T, Tominaga M. (2008) Drosophila Painless is a Ca2+ -Requiring Channels Activated by Noxiuos Heat. Journal of Neuroscience. 28:9929-9938. # of test irritant flies total # of flies feeding # of sucrose only flies PI =