1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

89 Symposium Taste & Smell in Translation:Applications from Basic ResearchTASTE AND SMELL IN TRANSLATION: APPLICATIONSFROM BASIC RESEARCHMargolskee R.F. 1 , Reed R.R. 2 , Herz R. 3 , Breslin P. 4 1 Neuroscience,Mount Sinai School of Medicine, New York, NY; 2 Molecular Biology &Genetics, Johns Hopkins University, Baltimore, MD; 3 Psychology,Brown University, Providence, RI; 4 Monell Chemical Senses Center,Philadelphia, PAThe symposium is an exploration of recent key advances in thechemical senses of interest to industry scientists, and basic scientists.The speakers are internationally known experts with a clear view of thecutting edge and a broad perspective. Each will focus on two or a fewrecent advances in basic research that have potential applications. Forexample: Recent characterization of taste and olfactory receptormolecules provides opportunities for high throughput screening and forde-novo design of commercially important tastants and olfactants;Rigorous examination of the effects of fragrances on human mood andbehavior point to applications with a firmer scientific basis; and,Genetic differences in taste perception are now seen to affect foodpreferences. The speakers will take the time to explain the basic sciencebackground behind their examples for an audience that cannot be expertin all relevant areas. The audience will include industry scientists andpolicy makers as well as basic scientists and students new to the field,interested in applications of basic research. The symposium willconclude with a round-table discussion with audience participation. Ourgoal is to explore how collaborations between industry and academicscientists can benefit both, but particularly how basic-science expertisecan contribute. The speakers' broad areas of interest are: Tastemolecular biology (Margolskee); Olfaction molecular biology (Reed);Olfaction perception/ psycho-physics (Herz); Taste perception/ psychogenomics(Breslin). The symposium will be followed by a receptionwith buffet and cash bar: An opportunity for industry participants tonetwork and to interact one-on-one with the symposium speakers andother interested basic scientists.90 Symposium Structure/Function and Pharmacology ofGPCRTHE ORGANISATION AND MOLECULAR RELEVANCE OFGPCR QUATERNARY STRUCTUREMilligan G. 1 1 University of Galsgow, Glasgow, United KingdomIt is now widely accepted that rhodopsin-like G protein coupledreceptors (GPCRs) exist as dimers or higher-order oligomers.However, apart from rhodopsin in rod outer segments the organisationalstructure of other GPCRs remains unclear. We mapped sites ofinteraction between monomers of the α1β-adrenoceptor using fragmentsfrom the receptor comprising the N-terminal domain linked to varioustransmembrane domains and intracellular loop connections.Symmetrical interactions were shown for TM1 and TMIV. Thisresulted in a model in which a `daisy chain´ of monomers generates anoligomeric structure. Because two protein resonance energy transfertechniques are poorly suited to distinguish dimers from oligomers weemployed `three colour´ fluorescence resonance energy transferimaging in single cells. Co-expression of forms of the α1βadrenoceptorC-terminally tagged with CFP, YFP and a FRP producedsequential 3 protein FRET that was absent when α1β-adrenoceptor-YFPwas replaced with the non-fluorescent construct α1β-adrenoceptor-Y67C-YFP. Mutation of key hydrophobic residues in TM1 and TMIVresulted in a marked reduction in sequential 3 colour FRET suggestingan alteration in oligomeric organisation. The mutated α1β-adrenoceptorwas expressed as well as the wild type but only a fraction was able tobind the ligand [3H]prazosin and the mutant was unable to reach thecell surface because it did not become core-glycosylated. Notsurprisingly, the mutant was completely unable to signal in response toaddition of α1β-adrenoceptor agonists.91 Symposium Structure/Function and Pharmacology ofGPCRPHARMACOLOGY OF MOUSE OLFACTORY RECEPTORSTouhara K. 1 1 University of Tokyo, Chiba, JapanAn olfactory receptor (OR) possesses a broad but selective ligandspectrum. Functional experimental analysis and computational dockingsimulation allowed for identification of the odorant-binding site of amouse OR, suggesting the molecular basis of the structure-activity. Wealso identified amino acids that were involved in receptor dynamicsfrom an inactive to an active conformation and in coupling to Gproteins. These studies revealed molecular mechanisms underlyingodorant binding and subsequent G protein activation of an OR. Thepharmacology of an OR in the olfactory epithelium is thought to reflectthe pharmacological property of the corresponding glomerulus in theolfactory bulb. We generated mOR-EG-ires-gapEGFP transgenic miceand performed calcium imaging to examine the odorant receptive rangeof the glomerulus innervated by mOR-EG-expressing neurons. Therelative thresholds for various ligands in the mOR-EG-glomerulus weredifferent from those observed in isolated mOR-EG-expressing neurons,suggesting that an odorant-induced activity pattern created in theolfactory bulb in vivo is not exactly reflected by peripheral ORpharmacology. Studies on OR function in the aspect of pharmacology,biophysics, and physiology will be presented. [supported byPROBRAIN, Japan]92 Poster Chemosensory Coding and ClinicalOLFACTORY EVENT-RELATED POTENTIALS: HOW MANYSTIMULI DO WE REALLY NEED?Boesveldt S. 1 , Haehner A. 2 , Berendse H. 1 , Hummel T. 2 1 Neurology, VUUniv. Medical Center, Amsterdam, Netherlands; 2 Otorhinolaryngology,Univ. of Dresden Medical School, Dresden, GermanyObjective Measurement of chemosensory event-related potentials(CSERPs) is useful to quantify olfactory function in a relativelyunbiased manner. We investigated the influence of the number ofdelivered stimuli on amplitude and signal-to-noise (S/N) ratio ofCSERPs. Methods CSERPs from 20 normosmic subjects were obtainedin response to stimulation by two olfactory (H 2 S and phenylethylalcohol), and one trigeminal (CO 2 ) stimulant. For each of these odours,a series of 160 stimuli was delivered into the right nostril (stimulusduration 200 ms, mean ISI 30 s) using a constant-flow, air-dilutionolfactometer. Only artefact-free trials were used for analysis. For eachEEG recording site (Fz, Cz, Pz, C3, C4), peak-to-peak amplitudes P1N1and N1P2 were determined, as well as noise amplitude levels.Subsequently, S/N ratios were calculated. Results The S/N ratio forolfactory ERPs significantly improves up to 80 delivered stimuli. Theoptimal number of stimuli for trigeminal ERPs is slightly lower, i.e. 60stimuli. This result is mainly due to a reduction of the noise-level withincreasing numbers of responses averaged. Applying more stimuli haslittle additional effect on the S/N ratio due to a concomitant decrease inthe amplitude of the signal. Conclusion S/N ratio in olfactory ERPs isoptimal when using 80 consecutive stimuli, while 60 stimuli appear tobe optimal for CO 2 . This research was funded by Philip Morris USAInc. and supported by the Dutch Parkinson´s Disease Association.23