2009 Abstracts - Association for Chemoreception Sciences

#P147 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyExpression of an Inwardly-Rectifying Potassium Channel(ROMK) in Mouse Glial-like Taste CellsGennady Dvoryanchikov 1 , Michael Sinclair 2 , Nirupa Chaudhari 1,21Department of Physiology and Biophysics, University of MiamiMiller School of Medicine Miami, FL, USA, 2 Program inNeurosciences, University of Miami Miller School of MedicineMiami, FL, USACells in taste buds are closely packed with little extracellularspace. Tight junctions and other barriers further limitpermeability and may result in the buildup of K + during actionpotentials. In many tissues, inwardly-rectifying K channels suchas the Renal Outer Medullary K (ROMK) channel help toredistribute K + . ROMK is an inwardly rectifying ATP-sensitiveK channel, derived from the kir1.1 (kcnj1) gene. Using RT-PCR,we defined several splice variants of ROMK in mouse kidney, andreport here the expression of a single one of these splice variants,ROMK2, in a subset of mouse taste cells. Using qRT-PCR, wefound ROMK2 mRNA is expressed in taste buds in the followingorder of abundance: vallate > foliate >> palate >> fungiform.Immunocytochemistry revealed that the ROMK protein followsthe same pattern as mRNA, and is essentially undetectable infungiform taste buds. ROMK is localized to the apical tips of asubset of taste cells (~8.5+/-2.5 cells/vallate tastebud). Usingtissues from PLCb2-GFP and GAD-GFP transgenic mice, weshow that ROMK is not expressed in PLC 2-expressing typeII/Receptor cells or in GAD-expressing type III/Presynaptic cells.Thus, immunocytochemical data suggest that ROMK expressionis limited to a subset of glial-like type I cells. Single-cell RT-PCRconfirms this interpretation: ROMK2 mRNA was detected in23% of NTPDase2-expressing cells, but not in either PLCb2- orSNAP25-expressing taste cells. We propose that in taste buds,ROMK in supporting type I cells may serve a homeostaticfunction, excreting excess K + through the apical pore, andallowing excitable taste cells (types II and III) to maintain ahyperpolarized resting membrane potential.#P148 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyCortical Processing of Learned Aversive Odors in Awake RatsChien-Fu F Chen 1,3 , Donald A Wilson 1,21Nathan Kline Institute Orangeburg, NY, USA, 2 NYU School ofMedicine New York, NY, USA, 3 The University of OklahomaNorman, OK, USAMost naturally occurring odors are complex mixtures. Thesemixtures are hypothesized to be synthesized into odor objectsthrough activity of olfactory cortical circuits. Work inanesthetized rats has demonstrated that neurons in the anteriorpiriform cortex discriminate between mixtures and theirindividual components. Furthermore, it has been shown thatcortical odor processing is experience-dependent, with familiarityleading to enhanced discriminability of odorants. However, thereis still limited data on cortical processing of odors in awakeanimals. The present experiment had two primary goals. First,compare activity of neurons in the anterior piriform cortex ofawake rats to those in anesthetized rats in response to complexmixtures and second, examine how aversive conditioning canaffect that processing. Long-Evans rats were chronicallyimplanted with movable wire bundles aimed at the anteriorpiriform cortex. Responses of single-units to individual odorantsand mixtures were tested. Odors were randomly presented fromthe top of the recording chamber to mimic natural odor plumes.Following several days of recording, one odor was chosen as theconditioning odor in an odor aversion conditioningparadigm. Unit data was recorded during the conditioning trialsand for several days post-training. The electrode was moved overtime to sample additional cells. Preliminary results (n = 148 units)suggest that 35% of the units responded to any one odor or odormixture, which is comparable to response rates in anesthetizedrats. Individual cells showed excellent discrimination of odors,including mixtures overlapping by as much as 90%. Finally, odoraversion appeared to enhance selectivity of anterior piriformcortex neuron ensembles, potentially enhancing thediscriminability of learned aversive odors.#P149 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyStrategy for recombinant expression of functionalN-terminal domain of human T1R3 taste receptorproduced in Escherichia coliElodie Maîtrepierre, Maud Sigoillot, Loïc BriandUMR 1129 INRA-ENESAD-UB FLAVIC Dijon, FranceThe sweet taste receptor is a heterodimer composed of twosubunits called T1R2 and T1R3. Each subunit belongs to the classC of G protein-coupled receptors (GPCRs) and is constituted bya large extracellular N-terminal domain (NTD) linked to thetransmembrane domain by a cystein-rich region. T1R2 and T1R3NTDs are both able to bind natural sugars and some sweeteners(sucralose) with distinct affinities and undergo ligand-dependentconformational change. However, the relative contribution of thetwo subunits to the heterodimeric receptor function remainslargely unknown. To study the binding specificity of each subunitusing biochemical and structural approaches, a large amount ofpurified NTDs is suitable. Here, we report the production offunctional human T1R3 NTD from insoluble aggregated protein(inclusion bodies) expressed in high level in Escherichia coli.Transferring this protein into its native state by in vitro refoldingrequires screening to find buffer conditions and suitable additives.We established a factorial screen to detect folded functional T1R3NTD based on intrinsic tryptophan fluorescence quenching bysucralose. From the screen, we successively identified positivesynergistic interactions between additives on refolding of T1R3NTD. The soluble protein was then purified and characterized.Fluorescence and circular dichroism spectroscopy demonstratedthat T1R3 NTD is properly refolded and able to bind saccharidecompounds with physiological relevant affinities. To furthervalidate our expression strategy, we introduced single amino acidchanges in the predicted binding site using site-directedmutagenesis. The described production procedure in highquantity should be useful to perform structural and functionalstudies of human T1R3 and other T1R ligand-binding domains.P O S T E R SAbstracts | 73