2009 Abstracts - Association for Chemoreception Sciences

subject to native regulatory mechanisms. Here we presentrecordings of TRPM5 currents from native rat posterior TRCsusing standard patch clamp techniques with cesium electrodes toblock potassium current. Using flash photolysis (with caged-IP 3or caged Ca 2+ ) or ionomycin (a calcium ionophore), a TRPM5-like current was consistently evoked in half of tested TRCs. Thecurrent displayed strong desensitization, rectification, dependenceon extracellular sodium, and was unaffected by a TRPM4 channelblocker. Additionally, the tastants cycloheximide or SC45647(each at 100 M) were demonstrated to evoke highly similarcurrent in a subpopulation of TRCs. As expected, the IP 3 -receptor blockers, 2-APB and heparin, and thapsigargin, aninhibitor of intracellular calcium stores, diminished the activationand amplitude of the TRPM5 current. Quinine, reported toinhibit TRPM5 currents, similarly had an inhibitory action on thiscurrent. Further the current was modulated by application of thelipid signaling agent PIP 2 which also promoted bitter and sweettastant-elicited TRPM5 responses. The current could also beevoked by arachidonic acid (AA) further suggesting itsmodulation by lipid regulators. Preliminary data from HEK293cells expressing mouse trpm5 supports our results from nativeTRCs with evoked currents displaying similar activation anddesensitization kinetics. Collectively, these data support thenotion that we are recording native TRPM5 currents from asubset of TRCs.#P167 Poster session IV: Chemosensory transductionand perireceptor eventsNovel Insights into Odorant Recognition: A Computationaland Functional Analysis of Ligand Binding to the HumanOlfactory Receptor OR2AG1Lian Gelis 1 , Steffen Wolf 2 , Klaus Gerwert 2 , Hanns Hatt 1 ,Eva M. Neuhaus 11Department of Cellular Physiology, Ruhr-University BochumBochum, Germany, 2 Department of Biophysics, Ruhr-UniversityBochum Bochum, Germany#P168 Poster session IV: Chemosensory transductionand perireceptor eventsOlfactory Neuron Response Statistics: a Cross Species AnalysisRafi Haddad 1,2 , David Harel 1 , Noam Sobel 21Department of Computer Science and Applied MathematicsRehovot, Israel, 2 Department of Neurobiology, the WeizmannInstitute of Science Rehovot, IsraelAlthough the olfactory systems of different species shareremarkable organizational similarities, there are also markeddifferences between them, most notably the number of receptortypes and their response dynamics. The reasons for, andimplications of these differences, are largely unknown. Here weanalyzed a large set of previously published olfactory neuralresponse data from different olfactory neurons in six differentspecies. We found that whereas species with a small number ofolfactory receptor (OR) types have more broadly tuned responsecurves, species with larger number of OR types tend to have morenarrowly tuned response curves. Consistent with this, specieswith larger number of OR types were better at discrimination ofenantiomers. Comparing the response spectra of all olfactoryneurons from the different species, we found that only a small setof olfactory neurons had similar response spectra. This lack offunctional conservation is consistent with genetic analysis, thattogether suggest each species may have olfactory receptors thatare optimized for its own ecological niche. Despite thesedifferences, we found that in all species tested, and across 12unrelated reported studies, the primary axis of the olfactoryneuronal space was the “total neuronal response” elicited bythe odors. In other words, summing the neural response elicitedby an ensemble of olfactory neurons to a specific odor accountsfor a large portion of the neural response variability. Critically,this primary axis of neural space was significantly correlated tothe odor preferences of rats, mice and drosophila. We thereforesuggest that the primary axis of neural response is related toodor hedonics.P O S T E R SThe process of odor perception begins with the binding ofodorant molecules by specific odorant receptors (ORs). So far,only few ligand-OR interaction pairs have been characterized andmost characterized receptors show a rather broad tuning anddetect multiple, chemically similar odorants. Due to the limitedknowledge on receptor – odorant interaction, the moleculardetails of ligand specificities of ORs are not well understood.Highly variable residues in the transmembrane domains III, IV,and V were postulated to form the basis for ligand specificity. Togain more insight into the molecular basis of odor recognition, weinvestigated ligand-receptor interactions for the human olfactoryreceptor OR2AG1 by combining dynamic homology modelingand ligand docking with functional analysis and site-directedmutagenesis. Basing on the rhodopsin crystal structure, wepredicted the protein structure of OR2AG1 by homologymodeling. The model was further subjected to free moleculardynamics simulations in an explicit membrane/solventenvironment with various odorants introduced into the putativebinding site. To verify the theoretical model experimentally wemutated amino acids predicted to be involved in ligand bindingand compared activation of receptor variants heterologouslyexpressed in Hana3a cells by Ca 2+ -imaging. By combiningtheoretical and experimental techniques it was possible tocharacterize ligand binding of OR2AG1 on the atomic scale.#P169 Poster session IV: Chemosensory transductionand perireceptor eventsDeterminants of Agonist Sensitivity in an Insect OlfactoryReceptorAndrew S. Nichols, Charles W. LuetjeMolecular and Cellular Pharmacology, University of Miami,Miller School of Medicine Miami, FL, USATo identify functionally important structural features ofDrosophila olfactory receptors (DmOrs), we expressed DmOrsOr35a, Or85a, and Or85b in Xenopus oocytes (each with Or83b),and screened for susceptibility to methanethiosulfonate (MTS)reagents. These reagents may alter receptor function by covalentlyattaching to accessible cysteines, thus providing an approach forstructural studies. One receptor, Or85b, was partially inhibited byMTSES (ES). We mutated each of the five cysteine residues (C124,C146, C208, C278, and C311) to serine and showed that onlymutant C146S failed to be inhibited by ES, suggesting C146 as thesite of ES covalent attachment and receptor inhibition.Comparison of current-voltage relationships before and after EStreatment indicated no effect on the ionic permeability propertiesof the receptor, suggesting that ES is not acting at the channelpore. Increasing agonist concentration reduced the inhibitoryability of ES, suggesting that ES was shifting the dose-responseAbstracts | 79