Download - Lecture 20 : Olfaction II
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Lecture 20 : Olfaction II Lecture 20 : Olfaction II
11/11/09
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For next weekFor next week
See web site Background - 1 pg
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TopicsTopics
1. Diversity of ORsWhich receptors detect which odorant?
2. Olfactory signal transductionHow does it compare to visual
transduction?
3. Multiple olfactory organsNose vs VNO : How do the receptors
compare?
4. Signal transductionHow do pathways compare?
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How do we understand How do we understand receptor specificity?receptor specificity?
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Functional response of rat Functional response of rat I7 receptorI7 receptor
I substitutionsII chain lengthIII/IV degree satIII/V side chain
ResponseR>G>B
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Functional groupFunctional groupCompare response of control cells vs ones containing I7 receptor
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Length and saturationLength and saturation
C8 is best though receptor responds to others too
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Effect of substitutionsEffect of substitutions
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Expressing olfactory Expressing olfactory receptorsreceptors
Ways to express genesMake transgenic animalsExpress in Xenopus oocytesIdeal to do in cell culture
Ways to probeIntracellular recording of one cellPull off patch of membraneCa+2 imaging
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Matsunami’s lab - cell Matsunami’s lab - cell cultureculture
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Cofactors for OR Cofactors for OR expressionexpression
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Olfactory signal Olfactory signal transductiontransduction
Odorant inactivates receptor
activates Golf
activates ACmakes cAMP
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Monitor cell response Monitor cell response with luciferasewith luciferase
CRE luciferase
Odorant in -> cAMP -> luciferase + luciferin = LIGHT
luciferin
CRE = cAMP response element
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Screen panel of 96 Screen panel of 96 receptorsreceptors
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Screen panel of 96 Screen panel of 96 receptorsreceptors
Flow reactant 1 and determine which receptors respond
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Screen panel of 96 Screen panel of 96 receptorsreceptors
Flow reactant 2 and determine which receptors respond
Map out response space
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OR responseOR response
Map out response to range of chemicals and odorant levels
Different receptors respond to different ligands
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Variation in human Variation in human sensitivitiessensitivities
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Human perceptual Human perceptual variationvariation
Androstenone is odorous steroidDerived from testosterone
Human response variesOffensive = Sweat / urinePleasant = sweet, floralOdorless
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OR7D4 receptor is sensitive OR7D4 receptor is sensitive for for
androstenone/androstadienonandrostenone/androstadienonee
Screened 335 OR’s (85% of known ORs!)
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Human allelic variation in Human allelic variation in OR7D4OR7D4
Search SNP database and seq in 391 test subjects - variation at 2 AAR88W T133M
Two common genotypes RT or WMNormal (RT) 0.786WM 0.157P79L 0.04S84N 0.013
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WM allele much less sensitive WM allele much less sensitive than RT allelethan RT allele
RT vs WM allele
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Common alleles affect Common alleles affect sensitivitysensitivity
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Rare alleles have opposite Rare alleles have opposite effectseffects
P79L not sensitive
S84N very sensitive
Chimp even more sensitive
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Genotype affects Genotype affects perceptionperception
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Human genotype affects Human genotype affects threshold sensitivitythreshold sensitivity
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Olfactory signal Olfactory signal transductiontransduction
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Olfactory signal Olfactory signal transductiontransduction
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Comparison of visual and Comparison of visual and olfactory activationolfactory activation
Vision Olfaction
G protein Gt Gs
Effector PDE AC
Effect on 2nd messenger
Destroys cGMP
Makes cAMP
2nd messenger binding
Comes off channel
Binds to channel
Ion channel response
Closes Opens
Neural response Hyperpolarize
Depolarize
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Comparison of visual and Comparison of visual and olfactory activationolfactory activation
Vision Olfaction
G protein GNAT1/2 GNAs,olf
Effector PDE Adenylate cyclase
2nd messenger cGMP cAMP
Ion channel CNGA1/3CNGB1/3
CNGA2/4CNGB1b
Ion channel response to signal
Closes, Ca2+ Opens, Na+ Ca2+
Neural response Hyperpolarize Depolarize
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Olfactory signal recoveryOlfactory signal recovery
1. OR* gets phophorylated and binds arrestin
2. PDE turns cAMP into AMP
This decreases cAMP binding to channel
Channel closes
#1
#2
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Olfactory signal recoveryOlfactory signal recovery
Adaptation
CaM-Ca+2 binds to channel and decreases cAMP binding
CaM-kinase phosphorylates PDE which acts faster
CaM kinase phosphorylates AC which acts slower
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Phototransduction Phototransduction recoveryrecovery
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Comparison of visual and Comparison of visual and olfactory deactivationolfactory deactivation
Vision Olfaction
Receptor phosphorylation
GRK GRK -
Receptor shut down Arrestin -arrestin
Calcium levelsCa-calmodulin binding
LowDecreased
HighIncreased
Channel binding cGMP binding cAMP binding
Effector offset Guanylate cyclase turns on GMP-cGMP
Phosphodiesterase turns on cAMP-AMP
Effector offset Adenylate cyclase slows down
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Multiple olfactory cuesMultiple olfactory cues
Need to detect general odors Food, environment
Species specific odorsPheromone =
pherein (to carry or transfer) +horman (to excite)
Volatile (broadcast info) or non-volatile (individual specific)
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Organs for detecting Organs for detecting compoundscompounds
Olfactory Vomeronasal Trigeminal
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Multiple olfactory organsMultiple olfactory organs
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Mouse olfactionMouse olfaction
Main olfactory epithelium (MOE)
Expresses olfactory receptors OR (1300) and Trace amine associated receptors TAAR
Project to main olfactory bulb (MOB)
Brennan and Zufall 2006
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Mouse olfactionMouse olfaction
Vomeronasal organ (VNO)
Expresses two types of receptors: V1R (137) and V2R (60)
Project to Accessory olfactory bulb (AOB)
Brennan and Zufall 2006
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VNOVNO
Two kinds of receptors which differ in location and projection
Response to putative pheremones
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Comparison in Comparison in rodentsrodents
Olfaction VNO1 VNO2
Location Olfactory epithelium
Apical VNO
Proximal VNO
Cell type Cilia Microvilli Microvilli
Receptors
OR V1R V2R
# in rodents
1000 290 100
Binding site
In TM In TM N-terminus
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V2R’s similar to mGlu V2R’s similar to mGlu receptorsreceptors
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Diversity of GPCRsDiversity of GPCRs
Bargmann 2006
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Comparison of pathwaysComparison of pathwaysOlfaction VNO1 VNO2
Receptor OR V1R V2R
G protein Gs Gi2 Go
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GGGs stimulates adenylate cyclase
includes olfactory
Gi/Go Includes vision and taste
Gq
Activates PLC
OR
V2R
V1R
Vision/taste
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Comparison of pathwaysComparison of pathwaysOlfaction VNO1 VNO2
Receptor OR V1R V2R
G protein Gs Gi2 Go
Effector / activator
AC / cAMP ?? ??
Ion channel CNGA2CNGA4/CNGB1
CNGA4 + TRP2
CNGA4 + TRP2
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Comparison of pathwaysComparison of pathwaysOlfaction VNO1 VNO2
Receptor OR V1R V2R
G protein Gs Gi2 Go
Effector / activator
AC / cAMP ?? ??
Ion channel CNGA2CNGA4/CNGB1
CNGA4 + TRP2
CNGA4 + TRP2
Projection Olfactory bulb Accessory olf bulb - anterior
Accessory olf bulb - posterior
Projection Olfactory cortex Amygdala / hypothalamus
Amygdala / hypothalamus
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VNO mystery #1VNO mystery #1
Are vomeronasal receptors for species specific communication?
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OR OR phylogephyloge
nyny
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VNO receptorsVNO receptors
Species specific as if they evolved for intra-species communication
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Mystery #2Mystery #2
What is the evolutionary history of the VNO?
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Phylogeny of VNOPhylogeny of VNOHuman
Mouse
Bird
Reptile
Amph
Fish
VNO X
V1R in OE?
V1R, V2R in VNO
V1R,V2R in OE
?
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Fish nose - 2 naresFish nose - 2 nares
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Fish olfactory epithelium has Fish olfactory epithelium has cilia (OR) and microvilli cilia (OR) and microvilli
(V1R, V2R)(V1R, V2R)
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Mystery #3 - Do humans Mystery #3 - Do humans have a VNO?have a VNO?
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AnatomyAnatomy
Structure is present in embryos
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CellularCellular
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NeuralNeural
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ElectricalElectrical
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Best case, worst caseBest case, worst case
Anatomical - structure exists Cellular - no sensory neurons Gene exp - no known V1R/V2R expressed in
VNO but maybe in OEHumans have lost TrpC2
Accessory bulb- doesn’t exist in humans Chemical response
- do record chemical dependent signal where VNO is
- is synchronization of menstrual cycles through chemical communication
Need more data
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New VNO receptors!