Lecture 20 : Olfaction II Lecture 20 : Olfaction II

11/11/09

For next weekFor next week

See web site Background - 1 pg

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?

How do we understand How do we understand receptor specificity?receptor specificity?

Functional response of rat Functional response of rat I7 receptorI7 receptor

I substitutionsII chain lengthIII/IV degree satIII/V side chain

ResponseR>G>B

Functional groupFunctional groupCompare response of control cells vs ones containing I7 receptor

Length and saturationLength and saturation

C8 is best though receptor responds to others too

Effect of substitutionsEffect of substitutions

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

Matsunami’s lab - cell Matsunami’s lab - cell cultureculture

Cofactors for OR Cofactors for OR expressionexpression

Olfactory signal Olfactory signal transductiontransduction

Odorant inactivates receptor

activates Golf

activates ACmakes cAMP

Monitor cell response Monitor cell response with luciferasewith luciferase

CRE luciferase

Odorant in -> cAMP -> luciferase + luciferin = LIGHT

luciferin

CRE = cAMP response element

Screen panel of 96 Screen panel of 96 receptorsreceptors

Screen panel of 96 Screen panel of 96 receptorsreceptors

Flow reactant 1 and determine which receptors respond

Screen panel of 96 Screen panel of 96 receptorsreceptors

Flow reactant 2 and determine which receptors respond

Map out response space

OR responseOR response

Map out response to range of chemicals and odorant levels

Different receptors respond to different ligands

Variation in human Variation in human sensitivitiessensitivities

Human perceptual Human perceptual variationvariation

Androstenone is odorous steroidDerived from testosterone

Human response variesOffensive = Sweat / urinePleasant = sweet, floralOdorless

OR7D4 receptor is sensitive OR7D4 receptor is sensitive for for

androstenone/androstadienonandrostenone/androstadienonee

Screened 335 OR’s (85% of known ORs!)

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

WM allele much less sensitive WM allele much less sensitive than RT allelethan RT allele

RT vs WM allele

Common alleles affect Common alleles affect sensitivitysensitivity

Rare alleles have opposite Rare alleles have opposite effectseffects

P79L not sensitive

S84N very sensitive

Chimp even more sensitive

Genotype affects Genotype affects perceptionperception

Human genotype affects Human genotype affects threshold sensitivitythreshold sensitivity

Olfactory signal Olfactory signal transductiontransduction

Olfactory signal Olfactory signal transductiontransduction

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

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

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

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

Phototransduction Phototransduction recoveryrecovery

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

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)

Organs for detecting Organs for detecting compoundscompounds

Olfactory Vomeronasal Trigeminal

Multiple olfactory organsMultiple olfactory organs

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

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

VNOVNO

Two kinds of receptors which differ in location and projection

Response to putative pheremones

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

V2R’s similar to mGlu V2R’s similar to mGlu receptorsreceptors

Diversity of GPCRsDiversity of GPCRs

Bargmann 2006

Comparison of pathwaysComparison of pathwaysOlfaction VNO1 VNO2

Receptor OR V1R V2R

G protein Gs Gi2 Go

GGGs stimulates adenylate cyclase

includes olfactory

Gi/Go Includes vision and taste

Gq

Activates PLC

OR

V2R

V1R

Vision/taste

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

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

VNO mystery #1VNO mystery #1

Are vomeronasal receptors for species specific communication?

OR OR phylogephyloge

nyny

VNO receptorsVNO receptors

Species specific as if they evolved for intra-species communication

Mystery #2Mystery #2

What is the evolutionary history of the VNO?

Phylogeny of VNOPhylogeny of VNOHuman

Mouse

Bird

Reptile

Amph

Fish

VNO X

V1R in OE?

V1R, V2R in VNO

V1R,V2R in OE

?

Fish nose - 2 naresFish nose - 2 nares

Fish olfactory epithelium has Fish olfactory epithelium has cilia (OR) and microvilli cilia (OR) and microvilli

(V1R, V2R)(V1R, V2R)

Mystery #3 - Do humans Mystery #3 - Do humans have a VNO?have a VNO?

AnatomyAnatomy

Structure is present in embryos

CellularCellular

NeuralNeural

ElectricalElectrical

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

New VNO receptors!