Transcript Genomics of sensory systems

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
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Idea
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Taste I
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Taste II
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Project
Backgnd
Unusual
senses
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25
Help (no
class)
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27
Thanks- giving
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29
30 Gains
and
losses
Dec 1
2 Prelim
results
Summary
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5
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Present1
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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