c 4 phototransduction

8/21/2019 C 4 Phototransduction

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Phototransduction from frogs to flies

Modeling signal transduction


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Eye as an evolutionary challenge

To suppose that the eye, with all its inimitable

contrivances , could have been formed by natural

selection, seems, I freely confess, absurd in the highest

possible degree. Yet reason tells me,

C. Darwin, The Origin of Species:

Darwin goes on to describe how eye may have evolved

through accumulation of gradual improvements

A number of different designs exist:e.g. vertebrate, molluskan or jellyfish camera eyes

or insect compound eye

The eye may have evolved independently 20 times!!

(read Cells, Embryos and Evolution by Gerhart&Kirschner)


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Copepod's 2 lens telescope

Trilobite fossil 500 million years

Black antHouse Fly

Scallop

Octopus

Cuttlefish

See Gerhart and Kirschener Cells Embr os and Evolution

Eyes are present in most metazoan phyla: how did they evolve?


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The grand challenge:

What can we learn from the similarities and differences

in the architecture/anatomy, development and

molecular machinery of eyes (or light sensitive cells) indifferent species?

To understand the evolutionary processes thatunderlie the appearance of a complex organ (an eye).

To what extent is the similarity between different

eyes (e.g. vertebrate and mollusk) is due to common

ancestry or is the result of evolutionary convergencedue to physical constraints?

BUTbefore we can address the question of evolution

we need first to learn how it works


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More broadly:

Molecular pathway(s) of phototransductionare similar to many other signaling pathways:

olfaction, taste, etc

Comparative Systems Biology


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Modeling

molecular mechanisms ofphoto-transduction

Case studies: 1) vertebrate and 2) insect

Phototransduction as a model signaling system


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Vertebrate Rods and Cones


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Vertebrate photoreceptor cell

hv

Light reduces the

dark current into the cell

Na+ Na+, Ca2+100mm

Rod

Outer

Segment

(2000Discs)

Discs

Rhodopsin


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Turtle Cone

Turtle Rod: a flash response family shown

on different time scales

Intracellular Voltage in Rods and Cones


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Measuring currents


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Patch clamp


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Single Photon Response


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Patch clamp recordings


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K

K

Na, Ca

Na, Ca

K

K KK

Na

Na Na

Na

cGMP

Ca,KCa,K

K K

cGMP

K

In the dark a standingcurrent circulatesthrough the rod.

Light shuts off theinflux of current into

the outer segment, while Kcontinues to exit the innersegment causing the cell

to hyperpolarize.

Na,Ca channels of outer segment need cGMP to stay open

Electrophysiology of Rods

ATP drivenNa/K pump


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Rhodopsin

undergoes light-induced

conformational change

opsin, a membrane proteinwith 7 transmembrane

segments

chromophore,11-cis retinal

+

Light detector protein: Rhodopsin


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cis/trans retinal transition


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cytoplasmic side

Light-induced conformational transition


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Rhodopsin is a G-Protein coupled receptor


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b2adrenergic receptor

Rhodopsin

cytoplasmic end

Seven-Helix

G-protein-

Coupled

Receptorsform a large

class(5% C. elegans

Genome !)

Rh d i i G P t i l d t


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Rhodopsin light

Rh*

Tabg Ta + TbgGDP GTP

about109rhodopsinmolecules and about108 G-protein moleculesper rod outer segment (ROS)

photoactivated rhodopsin

(Rh*) forms in about 1 ms

and serially activates 100 to

1000 Transducin moleculesper second

Transducinis a

heterotrimeric

G protein

specific to vision

Rhodopsin is a G-Protein coupled receptor


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gb

a

guanine nucleotide

binding site onasubunit

a subunit: 35-45 kDbsubunit: 35-40 kDg subunit: 6-12 kD

At least 15 different genes forasubunit and several differentgenes for b andgsubunits

G- proteins


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extracellularcell membrane

gbaGDPG protein coupled

receptor (GPCR)

Resting state:

(G protein off)

gb

aGDP

receptor activated byaction of external

signalBinding of activated

receptor to G protein

opens guanine nucleotide

binding pocket onasubunit

1.Steps in activation


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Theaand bg subunitsdissociate from activated

GPCR

bg

GDP

aGTP

gba

*GTP

2. GDP -- GTP exchange at nucleotidebinding site activates the G protein

gba*

GTP

3.

Activated a subunitstimulates effectorproteins: enzymes or

ion channels


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Rhodopsin light

Rh*

Tabg-GDP Ta-GTP+ Tbg

PDE PDE*

cGMP GMP

CNG Channels: OPEN CLOSED

Effector


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Guanylate Cyclase

GTP cGMP + PPi GMP

Phosphodiesterase

Where did cGMP come from?


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Enzymes:

Phosphodiesterase (PDE)

Adenylyl cyclase

Phospholipase C

hydrolyzes cyclicnucleotide;

e.g. cGMP GMP

synthesizes cAMPfrom ATP

hydrolyzes PIP2to produceIP3and diacylglycerol (DAG)

Ion channels: e.g. K, Ca and Na channels

G Protein Effectors Include

Shut off


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Rhodopsin light

Rh*

Tabg-GDP Ta-GTP+ TbgPDE PDE*

cGMP GMP


Next question:How are the activated

intermediates shut off?

Shut-off


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Rhodopsin inactivation


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gba

*GTP g

baGDP Pi+

In Time

gba

GDP

Taandbgre-associate

Ta-GDPdissociates from PDE*

which re-inhibits PDE*

Transducin is inactivated by the aintrinsic GTPaseactivity which hydrolyzes GTP to GDP

Accelerated

byPDE*

Th i t i i GTP ti it f th b it


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gb

a*GTP

The intrinsic GTPase activity of the asubunitis regulated by a GAP (GTPase accelerating protein)

RGS-9

gbaGDPPi

+

Ta*shut off is acceleratedby action of RGS-9

Regulator of G-protein signaling


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This is accelerated by low Ca (feedback signal!) that

stimulates Guanylate Cyclase

GTP cGMP + PPi

Recovery to the resting state requires resynthesis

of the cGMP that had been lost to hydrolysis.


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K

K

Na, Ca

Na, Ca

K

K KK

Na

Na Na

Na

cGMP

Ca,KCa,K

K K

cGMP

K

channel closure shuts off Cainflux, while efflux by

Na:Ca,K exchange

continues and internal Ca

declines. The fall in [Ca] is the

[Ca] feedback signal.

Ca- the second 2ndmessenger


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Ca2+

Regulation of Guanylate Cyclase

Negative feedback


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Signaling

cascade

Alberts et al, Mol Bio of the Cell

Converts a microscopic stimulus

activation of a single molecule

-into a macroscopicresponse

li h

Phototransduction Cascade


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Rhodopsin

light

Rh*

cGMP GMP


1st Stage of amplification:

200 - 1000 G*per Rh*

2nd Stage amplification:

each PDE* hydrolyzes~ 100 cGMP molecules.

Gabg- GDP G*a- GTP+ Gbg

Phosphodiesterase (PDE) PDE*

Total gain:

2 x 10

5

10

6

cGMP / Rh*

channel closure

generates

electrical signal

Phototransduction Cascade

as an enzymatic amplifier

GC*

GTP


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supporting cellolfactory knob

cell body

tight junction

mucus filmolfactory cilium

receptor dendrite

basement membraneaxon

Olfaction:

Odor Olfactory receptor


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Olfactory

receptor

Odor

ligand

R*

cAMP AMP

CNG Channels: OPEN

Gabg- GDP G*a- GTP+ Gbg

AC*

PDE*

DEPOLARIZATION

OF THE CELL

Olfactory receptor

cascade

ATP

AC Adenylate cyclase

R

I t b t Ph t t d ti


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Rhodopsin light

Rh*

Gq- GDP Gqa- GTP+ Gqbg

Phospholipase C PLC*

Trp Channels: CLOSED OPEN

PIP2 IP3 & DAG( phosphoinositol 3,4 bisphosphate )

?

Invertebrate Phototransduction

Cascade


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Enzymatic amplifier modules

G

G*

Rh* PDE*

cGMP

GTP GMP

GC PDE*

1ststage:

G-protein (Transducin)

activation and deactivation

2ndstage:

cGMP hydrolysisand synthesis

GTP

GDP

GTP

GDP Pi

Note: [GTP] / [GDP] & [GMP] acts a metabolic power supply


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More examples of amp modules

G

G*

GEF GAP

GTP

GDP

GTP

GDP Pi S

SP

Kinase Phosphotase

ATP

ADP

Pi

Small GTPases:

Ras, Rho, Rab, Rap, Reb,

cGMP

GTPGMP

ReceptorGuanylate

CyclasePDE5

Nitric

Oxide

Vasodilation

Viagra


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General push-pull amplifier circuit

Xa

Xc*

Eact Ede-act

d

dtX k X E k X Eact de act [ *] [ ][ ] ' [ *][ ]

[ *] [ ][ ] ' [ ]

[ ]X k Ek E k E

Xact

act de act

tot+

Steady state:

c

a

b

a+b c

Power supply:

Note: [X*]/[X] = e-bDE

because the system is held out of equilibrium byfixed [c]/[a] maintained by metabolism

maintainshigh [c]/[a]


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Amplifier gain and time constant

d

dtX X k X Etot act [ *] [ *] [ ] [ ]+

1

Small change in [X*] induced by a small change in[Eact]:

Linear response (in the Fourier domain):

[ *]( )

[ ]( )

[ ]X

E

k X

iact

+1

Time constant +

k E k E act de act[ ] ' [ ]

Static gaing k X [ ]

Note: g ~ the gain-bandwidth theorem !!

Linear analysis of vertebrate


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Linear analysis of vertebrate

phototransduction

Rh* deactivation + 2 amplifier modules

with negative feedback via [Ca++]

Good approximation to weak flash (single photon) response


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Linear analysis of the cascade

Ch cGMP

g g Rh

i i

G cGMP

G cGMP

* ~ [ ]( )*( )

( )( )

+ +1 1

PDE* ~ G*

Assuming stoichiometric processes ofPDE* and Ch* activation are fast :

Ch* ~ cGMP*

d

dt

G G g Rh

d

dtcGMP cGMP g G

G G G

cGMP cGMP cGMP

* * *

[ ] [ ] *

+

1 1

1 1


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Hard and Soft parameters

Kinetic constants/affinities -- hard parameters

change on evolutionarytime scale

Enzyme concentrations -- soft parameters that

can be regulated by

the cell.

Golden rule of biological networks:anything worth regulating is regulated

+

k E k E act de act[ ] ' [ ]g k X [ ]e.g.


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General behavior

X

Y

g g

i in

n

*( )

*( )

...

( )...( )

+ +1

11 1

Impulse response in the time domain:

X t

Y

*( )

*( )0 {

g g tn

n

n

1

1

.....

at short times

at times timesg g ent Max

1.../

10

...*( )

*( )~

/

n

n

n

tX t

Yg

te

For:With n=3

- good fit to

single-photon

response


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Case of feedback

Ca Caddt

Ca Ca g cGMP [ ] [ ] [ ] +

+ gF[Ca]

G G

cGMP cGMP

d

dtG G g Rh

d

dtcGMP cGMP g G

* * *

[ ] [ ] *

+

via Ch*

feedback

cGMP

Rh

g g i

i i i g g

G cGMP Ca

G cGMP Ca Ca F

( )

*( )

( )

( )[( )( ) ]

+

+ + +

1

1 1 1


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Consequences of negative feedback

cGMP

Rh

g g i

i i i g g

G cGMP Ca

G cGMP Ca Ca F

( )

*( )

( )

( )[( )( ) ]

+

+ + +

1

1 1 1

Reduction of static gain: g g g g

g gG cGMP

G cGMP

Ca F

( )1

g gCa FCa cGMP

Ca cGMP

( )

2

4

Accelerated recovery:

Ringing:

Signal and Noise: How much gain is


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Signal and Noise: How much gain isenough?

Single photon signal:

I ~ 2 pA

I Dark~ 60 pAI /I Dark~ 3%}

Whats the dominant source of

background noise?

Thermal fluctuations?

CVCV V k T

V

V

k T

CVDark B

Dark

B

Dark

2

2

6

210

~

ROS capacitance C ~ 20pF

Negligible !

R h


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Reaction shot noise

d

dtX X X t* ( ,..) ( *,..) ( ) ++

Langevin noise

(i.e. Gaussian, White)

++

( ) ( ) ( ) ( )t t0

E.g. if- = 0 consider DX* produced over time Dt

# of molecules

z

+D

D D D

X dt

X X dt t X

*

( *) * [ ( )] *

2 2 2Poisson

process

Ch l i i


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Channel opening noise

d

dtCh cGMP Ch Ch t Ch* ([ ]) * ( ) +

g 1

ChD* =< Ch*> = Chg([cGMP]D)ChTot

( *)

( *)~

* *

/

*

*

Ch Ch Ch

Ch

Ch Ch

Dark

D

2

2 1 21 1

10

1%

> 4

Channel flicker noise:

Plus fluctuations of [cGMP]:

( *) ( [ ]

*

Ch Ch f cGMP D cGMP Ch2 2

+ / ) ( ) >

Note: this Poisson lawholds generally formass action

< 3% signal


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cGMP fluctuations

Note: voltage response is coherent over the whole

rod outer segment, hence must consider

the whole volume Vros ~ 104mm3

#cGMP = [cGMP] Vros ~ 10mM 104

mm3

~ 107

Negligible fluctuations

L lit f i l h t


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Locality of single photon response

hv

Na+, Ca2+100mm

cGMP

depletion

G* and PDE*

activity

l D m s s m

l m s s m

cGMP cGMP cGMP

G

~

~

m m

m m

~ . ~

. ~*

10 5 20

10 5 2

3 2 1

2 1


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Single photon response variability

Baylor-Riecke:

coefficient of

variation

[ ]~ .

/D D

D

I I

I

p p

p

2 1 2

0 25

What is the largest source of


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What is the largest source ofresponse variability?

Spontaneous activation G -> G* ??

< 10-7 s-1 Negligible even with 108 G molecules !

Variability of Rh* on- time Dt ??

DG* ~ kDt = Rh*1storder Poisson process:

( )D D

D

t t

t

2 2

21

Baylor& Reicke measured:

( ) ~.

D D

DI I

Ip p

p

2 2

205

Poisson process of order 20 !!!

M lti t d ti ti f Rh*


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Multistep deactivation of Rh*

Meta-Rhodopsin shut-off:


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ATP

Rh* Rh*-PO4 Rh-PO4-Arrestin

Rhodopsin

Kinase Arrestin

(active) (inactive)

Arrestin is a 48kD

accessory protein

Rh Kinase is

turned on by

binding to Rh*

Meta-Rhodopsin shut-off:

S


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Summary:

What we have learned:

GPCR / cyclic nucleotide cascade

Enzymatic amplifier module

and linear response

Noise analysis

Tomorrow

from frog to fly and from linear to non-linear


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Acknowledgements

Anirvan Sengupta, (Rutgers)

Peter Detwiler (U. Washington)

Sharad Ramanathan (CGR/Lucent)

c 4 phototransduction

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