Phototransduction from frogs to flies

Modeling signal transduction

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)
Copepod's 2 lens telescope
Trilobite fossil 500 million years
Black ant
House Fly
Scallop
Octopus
Cuttlefish
See Gerhart and Kirschener, Cells, Embryos and Evolution
Eyes are present in most metazoan phyla: how did they evolve?

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) in
different species?
To understand the evolutionary processes that
underlie 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 convergence
due to physical constraints?
BUT before we can address the question of evolution
we need first to learn how it works
More broadly:
Molecular pathway(s) of phototransduction
are similar to many other signaling pathways:
olfaction, taste, etc


Comparative Systems Biology
Modeling
molecular mechanisms of
photo-transduction
Case studies: 1) vertebrate and 2) insect
Phototransduction as a model signaling system
Vertebrate Rods and Cones
Vertebrate photoreceptor cell
hv
Light reduces the
dark current into the cell
Na
+
Na
+
, Ca
2+

100m
Rod
Outer
Segment

(2000
Discs)
Discs
Rhodopsin
Turtle Cone
Turtle Rod: a flash response family shown
on different time scales

Intracellular Voltage in Rods and Cones
Measuring currents
Patch clamp
Single Photon Response
Patch clamp recordings
K
K
Na, Ca
Na, Ca
K
K K
K
Na
Na
Na
Na
cGMP
Ca,K Ca,K
K K
cGMP
K
In the dark a standing
current circulates
through the rod.
Light shuts off the
influx of current into
the outer segment, while K
continues to exit the inner
segment causing the cell
to hyperpolarize.
Na,Ca channels of outer segment need cGMP to stay open
Electrophysiology of Rods
ATP driven
Na/K pump
Rhodopsin
undergoes light-induced
conformational change
opsin, a membrane protein
with 7 transmembrane
segments
chromophore, 11-cis retinal
+
Light detector protein: Rhodopsin
cis/trans retinal transition
cytoplasmic side
Light-induced conformational transition
Rhodopsin is a G-Protein coupled receptor
|
2
adrenergic receptor
Rhodopsin
cytoplasmic end
Seven-Helix
G-protein-
Coupled
Receptors
form a large
class
(5% C. elegans
Genome !)
Rhodopsin
light
Rh*
To| To
-
+ T|
GDP GTP
about 10
9
rhodopsin
molecules and about
10
8
G-protein molecules
per rod outer segment (ROS)
photoactivated rhodopsin
(Rh*) forms in about 1 ms
and serially activates 100 to
1000 Transducin molecules
per second
Transducin is a
heterotrimeric
G protein
specific to vision
Rhodopsin is a G-Protein coupled receptor

|
o
guanine nucleotide
binding site on
o subunit
o subunit: 35-45 kD

| subunit: 35-40 kD
subunit: 6-12 kD
At least 15 different genes for
o subunit and several different
genes for | and subunits
G- proteins
extracellular
cell membrane

|
o
GDP
G protein coupled
receptor (GPCR)
Resting state:
(G protein off)

|
o
GDP
receptor activated by
action of external
signal
Binding of activated
receptor to G protein
opens guanine nucleotide
binding pocket on
o subunit
1.
Steps in activation
The o and | subunits
dissociate from activated
GPCR

|

GDP
o
GTP

|
o
*

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

|
o
*

GTP
3.
Activated o subunit
stimulates effector
proteins: enzymes or
ion channels
Rhodopsin
light
Rh*
To|-GDP To
-
-GTP + T|
PDE PDE*
cGMP GMP
CNG Channels: OPEN CLOSED
Effector
Guanylate Cyclase
GTP cGMP + PPi GMP
Phosphodiesterase
Where did cGMP come from?
Enzymes:
Phosphodiesterase (PDE)


Adenylyl cyclase


Phospholipase C
hydrolyzes cyclic
nucleotide;
e.g. cGMP GMP
synthesizes cAMP
from ATP
hydrolyzes PIP
2
to produce
IP
3
and diacylglycerol (DAG)
Ion channels:
e.g. K, Ca and Na channels
G Protein Effectors Include
Rhodopsin
light
Rh*
To|-GDP To
-
-GTP + T|
PDE PDE*
cGMP GMP
CNG Channels: OPEN CLOSED
Next question:
How are the activated
intermediates shut off?
Shut-off
Rhodopsin inactivation

|
o
*

GTP
|
o
GDP P
i

+
In Time

|
o
GDP
To and |
re-associate
To-GDP
dissociates from PDE*
which re-inhibits PDE*
Transducin is inactivated by the o intrinsic GTPase
activity which hydrolyzes GTP to GDP
Accelerated
by PDE*

|
o
*

GTP
The intrinsic GTPase activity of the o subunit
is regulated by a GAP (GTPase accelerating protein)
RGS-9

|
o
GDP
P
i

+
To
*

shut off is accelerated
by action of RGS-9
Regulator of G-protein signaling
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.

K
K
Na, Ca
Na, Ca
K
K K
K
Na
Na
Na
Na
cGMP
Ca,K Ca,K
K K
cGMP
K
channel closure shuts off Ca
influx, while efflux by
Na:Ca,K exchange
continues and internal Ca
declines. The fall in [Ca] is the
[Ca] feedback signal.
Ca- the second 2
nd
messenger
Ca
2+
Regulation of Guanylate Cyclase
Negative feedback
Signaling
cascade
Alberts et al, Mol Bio of the Cell
Converts a microscopic stimulus
activation of a single molecule
-into a macroscopic response
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.
G
o|
- GDP G*
o
- GTP + G
|
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
supporting cell
olfactory knob
cell body
tight junction
mucus film
olfactory cilium
receptor dendrite
basement membrane
axon
Olfaction:
Olfactory
receptor
Odor
ligand
R*
cAMP AMP
CNG Channels: OPEN
G
o|
- GDP G*
o
- GTP + G
|
AC*
PDE*
DEPOLARIZATION
OF THE CELL
Olfactory receptor
cascade

ATP
AC Adenylate cyclase
R
Rhodopsin
light
Rh*
G
q
- GDP
G
q
o
-
- GTP + G
q
|
Phospholipase C PLC*
Trp Channels: CLOSED OPEN
PIP2 IP3 & DAG
( phosphoinositol 3,4 bisphosphate )
?
Invertebrate Phototransduction
Cascade

Enzymatic amplifier modules
G
G*
Rh*
PDE*
cGMP
GTP
GMP
GC PDE*
1
st
stage:
G-protein (Transducin)
activation and deactivation
2
nd
stage:
cGMP hydrolysis
and synthesis
GTP
GDP
GTP
GDP
P
i

Note: [GTP] / [GDP] & [GMP] acts a metabolic power supply
More examples of amp modules
G
G*
GEF
GAP
GTP
GDP
GTP
GDP
P
i

S
S
P
Kinase
Phosphotase
ATP
ADP
P
i

Small GTPases:
Ras, Rho, Rab, Rap, Reb,
cGMP
GTP
GMP
Receptor
Guanylate
Cyclase
PDE5
Nitric
Oxide
Vasodilation
Viagra
General push-pull amplifier circuit
X
a

X
c
*
E
act

E
de-act

d
dt
X k X E k X E
act de act
[ *] [ ][ ] ' [ *][ ] =

[ *]
[ ]
[ ] ' [ ]
[ ] X
k E
k E k E
X
act
act de act
tot
=
+

Steady state:
c
a
b
a+b c
Power supply:
Note: [X*]/[X] = e
-|AE

because the system is held out of equilibrium by
fixed [c]/[a] maintained by metabolism
maintains high [c]/[a]
Amplifier gain and time constant
d
dt
X X k X E
tot act
o t o o [ *] [ *] [ ] [ ] + =
1
Small change in [X*] induced by a small change in [E
act
]:
Linear response (in the Fourier domain):
o e
o e
t
et
[ *]( )
[ ]( )
[ ] X
E
k X
i
act
=
+ 1
Time constant
t = +

k E k E
act de act
[ ] ' [ ]
Static gain g k X = t [ ]
Note: g ~ t the gain-bandwidth theorem !!
Linear analysis of vertebrate
phototransduction
Rh* deactivation + 2 amplifier modules
with negative feedback via [Ca
++
]
Good approximation to weak flash (single photon) response
Linear analysis of the cascade
o o e
o e
et et
Ch cGMP
g g Rh
i i
G cGMP
G cGMP
* ~ [ ]( )
*( )
( )( )
=
+ + 1 1
o PDE* ~ o G*
Assuming stoichiometric processes of
PDE* and Ch* activation are fast :
o Ch* ~ o cGMP*
d
dt
G G g Rh
d
dt
cGMP cGMP g G
G G G
cGMP cGMP cGMP
o t o t o
o t o t o
* * *
[ ] [ ] *
= +
=


1 1
1 1
Hard and Soft parameters
Kinetic constants/affinities -- hard parameters
change on evolutionary
time scale

Enzyme concentrations -- soft parameters that
can be regulated by
the cell.
Golden rule of biological networks:
anything worth regulating is regulated
t = +

k E k E
act de act
[ ] ' [ ]
g k X = t [ ]
e.g.
General behavior
o e
o e et et
X
Y
g g
i i
n
n
*( )
*( )
...
( )...( )
=
+ +
1
1
1 1
Impulse response in the time domain:
o
o
X t
Y
*( )
*( ) 0
=
{
g g
t
n
n
n
1
1
...
.. t t
at short times
at times times g g e
n
t
Max
1
...
/ t
t t t
o
o t
t
1
0
= = =

...
*( )
*( )
~
/
n
n
n
t
X t
Y
g
t
e
For:
With n=3
- good fit to
single-photon
response
Case of feedback
t o o o
Ca Ca
d
dt
Ca Ca g cGMP [ ] [ ] [ ] = +
+ g
F
o[Ca]
t o o o
t o o o
G G
cGMP cGMP
d
dt
G G g Rh
d
dt
cGMP cGMP g G
* * *
[ ] [ ] *
= +
=
via oCh*
feedback
o e
o e
et
et et et
cGMP
Rh
g g i
i i i g g
G cGMP Ca
G cGMP Ca Ca F
( )
*( )
( )
( )[( )( ) ]
=
+
+ + +
1
1 1 1
Consequences of negative feedback
o e
o e
et
et et et
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 g
G cGMP
G cGMP
Ca F

( ) 1
g g
Ca F
Ca cGMP
Ca cGMP
>
( ) t t
t t
2
4
Accelerated recovery:
Ringing:
Signal and Noise: How much gain is
enough?
Single photon signal:
oI ~ 2 pA
I
Dark
~ 60 pA
oI /I
Dark
~ 3%
}
Whats the dominant source of
background noise?
Thermal fluctuations?
CV
CV V k T
V
V
k T
CV
Dark B
Dark
B
Dark
2
2
6
2
10 = =

o
o
~
ROS capacitance C ~ 20pF
Negligible !
Reaction shot noise
d
dt
X X X t * ( ,.. ) ( *,..) ( ) = +
+
I I q
Langevin noise
(i.e. Gaussian, White)
< >= +
+
q q o ( ) ( ) ( ) ( ) t t 0 I I
E.g. if I
-
= 0 consider AX* produced over time At
# of molecules
< >=
< > < > =< >=< >
z
z
+
A I
A A A
X dt
X X dt t X
*
( *) * [ ( )] *

2 2 2
q
Poisson
process
Channel opening noise
d
dt
Ch cGMP Ch Ch t
Ch
* ([ ]) * ( ) = +

t q
1
Ch
D
* =< Ch*> = t
Ch
([cGMP]
D
)Ch
Tot
< >=< >=
< >
<
= =
( *)
( *)
~
* *
/
*
*
o
o
Ch Ch Ch
Ch
Ch
Ch
Dark
D
2
2 1 2
1 1
10
1%

>

4
Channel flicker noise:
Plus fluctuations of o[cGMP]:
< >= < ( *) ( [ ]
*
o t t o Ch Ch f cGMP
D cGMP Ch
2 2
+ / ) ( ) >
Note: this Poisson law
holds generally for
mass action
< 3% signal
cGMP fluctuations
Note: voltage response is coherent over the whole
rod outer segment, hence must consider
the whole volume V
ros
~ 10
4
m
3

#cGMP = [cGMP] V
ros
~ 10M 10
4
m
3
~ 10
7
Negligible fluctuations
Locality of single photon response
hv
Na
+
, Ca
2+

100m
cGMP
depletion
G* and PDE*
activity
l D m s s m
l m s s m
cGMP cGMP cGMP
G
~
~
t

~ . ~
. ~
*
10 5 20
10 5 2
3 2 1
2 1

Single photon response variability

Baylor-Riecke:

coefficient of
variation
< < > >
< >
[ ]
~ .
/
A A
A
I I
I
p p
p
2 1 2
0 25
What is the largest source of
response variability?
Spontaneous activation G -> G* ??
< 10
-7
s
-1
Negligible even with 10
8
G molecules !
Variability of Rh* on- time At ??
AG* ~ kAt <At> = t
Rh*
1
st
order Poisson process:

< > < >
< >
=
( ) A A
A
t t
t
2 2
2
1
Baylor& Reicke measured:

< > < >
< >
( )
~.
A A
A
I I
I
p p
p
2 2
2
05
Poisson process of order 20 !!!

Multistep deactivation of Rh*
ATP
Rh* Rh*-PO
4
Rh-PO
4
-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:
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
Acknowledgements
Anirvan Sengupta, (Rutgers)

Peter Detwiler (U. Washington)

Sharad Ramanathan (CGR/Lucent)