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Lecture 21 : Taste I - Receptors 11/16/09 Final project background - 1 pg What are you studying? What is known about it? Why is it interesting? What do you want to find out? What methods / approach are you using? List 4 references you are using Background This is due either 11/18 OR 11/23 Choose the date based on when you can make substantial progress to move your project forward S M T Novem ber W Th F S 4 5 6 7 8 9 10 11 Idea 12 13 14 15 16 Taste I 17 18 Taste II 19 20 21 22 23 Project Backgnd Unusual senses 24 25 Help (no class) 26 27 Thanks- giving 28 29 30 Gains and losses Dec 1 2 Prelim results Summary 3 5 6 7 Present1 8 9 Prjct due Present 2 4 Final projects 2 weeks to develop some preliminary results Detailed summary of one or two papers or Preliminary analysis of some molecules or … Difference between taste and smell Fewer receptors Diversity? Take it in or send it out Drosophila taste Sensory cells all over body Legs taste first Extend proboscis and taste Intake and taste with taste papillae on oral surface Proboscis extension Sensory bristles on feet Labellum of probuscis Easy behavioral test for preference and for taste sensitivity Gustatory hairs Single hair has multiple cell types 1 mechanoreceptor 2-4 chemoreceptors Fig 2.8 Multiple sensory responses Mechanoreception S neuron Sugar, amino acids L1 neuron Salt (NaCl) Anion receptor - fatty acids ?? Pollack and Balakrishnan 1997 Fly sensory perception Mechanoreception - ionotropic Salt - ionotropic Anion receptor - ionotropic S neuron - ionotropic Sugar gated ion channel Sucrose detection is ionotropic Search Drosophila genome Find 43 GPCRs No homology to known odorant receptors No homologs in vertebrates Highly divergent from each other 8-20% similar High divergence - only TM7 is similar Clyne Fig 1 Several clusters of splice variants All have same 7th TM but 1-6 are different Clyne et al 2000 fig 3 How could they test whether these are for taste? RT-PCR to show gene is expressed in labellum and not elsewhere All expressed in labellum suggesting are gustatory Scott et al found new genes and tested where all genes were expressed Took advantage of Gal4-UAS system of gene expression Dow, J. A. T. J Exp Biol 2007;210:1632-1640 Express Gal4 and UAS in separate lines and then cross them UAS sites upstream of report like GFP Gal4 expression driven in tissue of interest Different receptors in different cells Proboscis proboscis antennae In situ hybridization of mRNA Transgenic expression of Gal4 driven by gene specific promoter Proboscis Mouth parts Legs GFP driven by GR promoter Scott et al 2001 Receptors expressed in several important body parts Sugar GPCRs have been found Drosophila Gr64 family - 6 genes Knock out all Gr64 genes - Gr64 Lose sensitivity to nearly all sugars Proboscis extension reflex A new taste in Drosophila? Unique Drosophila receptors Taste receptors identified from screen Gr5a - sweet Gr66a - bitter E409 - New receptor Use calcium imaging to determine sensitivity : CO2 Use calcium imaging to determine sensitivity : CO2 Response of olfaction and taste to CO2 are opposite and independent Questions What is the receptor? Is it a new kind of GPCR? Is it unique to Drosophila? Questions 1. What are mammalian taste receptors? 2. What mechanisms do they work by? Five kinds of taste Sweet Sour Salty Bitter Umami MSG / AA present proteins (meat broth, cheese) and Chinese food Three kinds of taste papillae Fungiform contain only 1-2 buds Circumvallate may contain 1000’s Multiple taste receptor cells (50150) come together to make a taste bud Taste buds connect to cranial nerves Taste neurons do not project to brain Synapse on cranial nerves which carry the gustatory signal to brain Might make easier to regenerate? Record from taste buds Determine sensitivity Single taste bud can be sensitive to multiple taste modalities Modalities likely controlled by different cells and transduction mechanisms, but reside in same taste bud! Chandrashekar et al 2006 Papillae made up of many taste buds 1000 100s few Diversity of taste mechanisms Metabotropic Ionotropic Sense To detect Sweet Nutrients Bitter Harmful Umami Amino acids Salty Salt balance Sour Harmful No tongue map - all taste buds detect multiple signals Neural wiring alternatives Chandreshakar 2006 Attractant taste Sweet and umami are desired substances Sweet receptor responds to all sugars, artificial sweeteners Umami responds to L-amino acids Monosodium glutamate, aspartate Use of mouse mutants to determine taste genes Chandrashekar et al 2006 Use of mouse mutants to determine taste genes Umami=T1R1+T1 R3 Sweet=T1R2+T1R3 Both need T1R3 to form heterodimer receptor Chandrashekar et al 2006 Sweet and umami GPCRs Xu et al 2004 Venus fly trap binding site Bitter detection Detect a diverse array of chemicals Avoidance Don’t need to distinguish May require a larger family of receptors Use of mouse mutants to determine taste genes Sensitivity to bitter is a related group of genes Bitter=T2R Chandrashekar et al 2006 Use of mouse mutants to determine taste genes PLC-2 is effector TrpM5 is channel Shared by all 3 Chandrashekar et al 2006 Bitter, sweet and umami receptors activate G protein, gustducin which stimulates phospholipase C T1R1+T1R3 PLC Salty and sour detection are different pathway Pkd2l1 is candidate sour receptor Member of TRP channel family Mammalian receptor for CO2 Pkd2L1 containing cells contribute to CO2 sense Carbonic anhydrase 4 converts CO2 to HCO3 which Pkd2l cell detects Car4-/- does not affect sour detection BZA and DZA inhibit Car4 so decrease CO2 response in WT Each taste modality relies on different receptors CO2 and pH Salt detection likely a channel Membrane responds to Na+ gradient RT Nao K o Vm ln F Nai K i Taste modalities are wired so brain responds in desired way Sweet receptors wired for attraction; bitter receptors for avoidance
