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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
CNG Channels: OPEN CLOSED
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
CNG Channels: OPEN CLOSED
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)