1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

365 Poster Chemosensory Molecular Genetics andVNO/PheromonePROPERTIES OF THE SEVEN TRANSMEMBRANE COREDOMAINS OF THE HUMAN T1RSSainz E. 1 , Cavenagh M.M. 1 , Lopezjimenez N.D. 1 , Battey J.F. 1 , NorthupJ.K. 1 , Sullivan S.L. 1 1 National Institute on Deafness and OtherCommunication Disorders, National Institutes of Health (NIH),Rockville, MDThe human T1R family of taste receptors consists of three family CG-protein-coupled receptors that act as heterodimers to detect sweettastingcompounds and amino acids. Although recent experiments haveexamined the ligand binding properties of these receptors, little isknown about their signaling properties. Using a baculoviral system, weexpressed truncated forms of the T1R receptors, which have intactseven transmembrane core domains but lack extracellular domains.Receptor-enriched membranes were purified from infected insect cellsand used in in vitro reconstitution assays. We demonstrate that the coredomains of T1R1 and T1R2 when expressed alone displayed significantconstitutive activity, catalyzing by several fold the exchange of GDP forGTP on transducin, a close homolog of gustducin. The constitutiveactivity of either the T1R1 or T1R2 core domain was unaffected by theaddition of lactisole or cyclamate and did not require dimer formationwith their common partner, T1R3. These results indicate that the invitro reconstitution assay can be used to determine the G proteinselectivities of the T1Rs. Furthermore, the results are consistent with thesuggestion that in their native heterodimer configurations (T1R1/T1R3or T1R2/T1R3), T1R1 and T1R2 are the signaling components, and theT1R3 core domain contributes the allosteric ligand binding site. Thiswork was sponsored by the Divisions of Intramural Research of theNIDCD and NINDS, NIH.366 Poster Chemosensory Molecular Genetics andVNO/PheromoneEFFECT OF MAILLARD PEPTIDES (MPS) ON TRPV1VARIANT SALT TASTE RECEPTOR (TRPV1T)Rhyu M. 1 , Ogasawara M. 2 , Egi M. 2 , Phan T.T. 3 , Desimone J.A. 3 , HeckG.L. 3 , Lyall V. 3 1 Food Function Research Division, Korea FoodResearch Institute, Korea, Songnam-Si, Kyunggi-D0, South Korea;2 Food Creation Center, Kyowa Hakko Food Specialities Co., Ltd., Amimachi,Ibaraki Prefecture, Japan; 3 Physiology, Virginia CommonwealthUniversity, Richmond, VAProteolysis occurs during ripening of protein-rich foods. Theresulting protein hydrolysate plays an important role in enhancing theflavour and taste of food. We investigated the effect of the naturallyoccurring MPs fractionated by ultrafiltration (500-10,000 Da) frommature Korean soy sauce on human salt taste perception. MPs (0.05%)presented a significant salt-masking activity. To test if MPs modulatesalt taste by interacting with TRPV1t, we monitored benzamil (Bz)-insensitive chorda tympani (CT) taste nerve responses in rats and inwild type and TRPV1 knockout (KO) mice. In 100 mM NaCl + 5 µMBz, varying the concentration of MPs (0-1.5%) produced biphasic CTresponses in rat and wild type mouse. Between 0.1% and 0.5%concentration, MPs increased the Bz-insensitive NaCl CT response.Above 0.5%, MPs inhibited the response, and at 1.5% the response wasdecreased to baseline. SB-366791 (1 µM), a specific inhibitor ofTRPV1t, eliminated the constitutive Bz-insensitive NaCl CT responseand inhibited the response to 0.4% MPs by 60%. In the presence of0.4% MPs, raising the temperature from 23° to 42° increased the CTresponse by 116.6%. TRPV1 KO mice demonstrated no Bz-insensitiveNaCl CT response and no response to MPs above baseline. Weconclude that MPs modulate the Bz-insensitive NaCl CT response andhuman salt taste by interacting with the TRPV1t cation channel infungiform taste receptor cells. Supported by NIDCD grants DC-005981(VL) and DC-00122 (JAD).367 Poster Chemosensory Molecular Genetics andVNO/PheromoneEFFECT OF N-GERANYL CYCLOPROPYLCARBOXAMIDE(NGCC) ON TRPV1 VARIANT SALT TASTE RECEPTOR(TRPV1T)Dewis M.L. 1 , Desimone J.A. 2 , Phan T.T. 2 , Heck G.L. 2 , Lyall V. 21 Flavor Ingredients R&D, International Flavors & Fragrances, UnionBeach, NJ; 2 Physiology, Virginia Commonwealth University, Richmond,VAA compound synthesized by IFF, NGCC (1 µM), significantlyenhanced salt taste perception in human psychophysical studies. To testif NGCC modulates salt taste by interacting with TRPV1t, wemonitored benzamil (Bz)-insensitive chorda tympani (CT) taste nerveresponses in rats. In 100 mM NaCl + 5 µM Bz, varying theconcentration of NGCC (0-50 µM) produced biphasic CT responses.Between 0.25 µM and 2.5 µM, NGCC increased the Bz-insensitiveNaCl CT response and above 2.5 µM it was inhibitory. At 50 µMNGCC, the CT response was not different from baseline. SB-366791 (1µM), a specific inhibitor of TRPV1t, eliminated the constitutive BzinsensitiveNaCl CT response and inhibited the entire tonic response atNGCC concentrations between 0.25 µM and 50 µM. Increasing thetemperature between 23° and 55.5° produced a biphasic increase in theCT response with a maximum response around 43°. NGCC (2 µM)increased the CT response at all temperatures without a shift in thetemperature threshold of the response. In contrast, N-cyclopropyl E2Z6-nonadienamide, a chemically related compound, did not affecthuman salt taste and also demonstrated no effect on the Bz-insensitiveNaCl CT response at concentrations between 0.25 µM to 50 µM. Weconclude that NGCC produces its effect on human salt taste byspecifically interacting with TRPV1t in fungiform taste receptor cellsand by modulating the Bz-insensitive NaCl responses. Supported byNIDCD grants DC-005981 (VL) and DC-00122 (JAD).368 Poster Chemosensory Molecular Genetics andVNO/PheromoneEXPRESSION OF FATTY ACID-ACTIVATED G PROTEINCOUPLED RECEPTORS IN CHEMOSENSORY CELLSHansen D.R. 1 , McKenna L. 1 , Shah B.P. 1 , Gilbertson T.A. 1 1 Biology &The Center for Integrated BioSystems, Utah State University, Logan,UTFatty acids (FA) have been implicated as chemosensory cues thatconvey the taste and texture of fat, where they have been shown tointeract with either delayed rectifying K + (DRK) channels and/or the FAtransporter CD36 in taste receptor cells. Recently, a number of orphanG protein coupled receptors (GPCRs) have been shown to be activatedby free FA in a variety of cell types. Using RT-PCR on mRNA isolatedfrom several chemosensory cell types including taste buds, trigeminalneurons and an enteroendocrine cell line, we have probed for expressionof DRK channels, CD36, and the fatty acid-activated GPCRs: GPR40,GPR41, GPR43 and GPR120. While FA-sensitive DRKs are found inall three rat lingual taste buds, expression of CD36 appears limited tothe foliate and vallate papillae, which also expresses GPR40, GPR41and GPR43, but not GPR120. We are currently exploring the subtypesof cells within the taste bud which express each receptor type by singlecell PCR. Enteroendocrine cells, which also respond to dietary fat,express FA-sensitive DRK channels, GPR120, GPR40, GPR41,GPR43, but not CD36. Trigeminal neurons, which contribute to thetextural properties of fats, also express a variety of putative fatreceptors. In addition to a variety of FA-sensitive DRK channels andCD36, trigeminal neurons express several of the aforementioned FAactivatedGPCRs. Our laboratory is continuing to explore the functionalconsequences of the expression of this variety of different fatty acidresponsiveproteins. Supported by DK59611 and Utah Ag. Expt. Station#630 (TAG).92