ioo msc phototransduction and photopigments - microsoft powerpoint

Andrew Stockman

Phototransduction & Photopigments 1

Phototransduction and photopigments

Andrew StockmanMSc BIOLOGY OF VISION

Introduction to Visual Neuroscience

Background

Light 400 - 700 nm is important for visionThe retina is carpeted with light-

sensitive rods and cones

An inverted image is formed on the retina

Andrew Stockman


Rods and cones

Webvision

Human photoreceptors

RodsAchromatic night vision

ConesDaytime, achromatic and chromatic visiong

1 type

Middle-wavelength-sensitive (M) or

Long-wavelength-sensitive (L) or “red” cone

3 types

Rod

Short-wavelength-sensitive (S) or “blue” cone

sensitive (M) or “green” cone

Rod and cone distribution

0.3 mm of eccentricity is about 1 deg of visual angle

Rod density peaks at about 20 deg eccentricity

At night, you have to look away from things to see

them in more detail

Andrew Stockman


During the day, you have to look at things directly to see them in detail

Cones peak at the centre of vision at 0 deg

Original photograph

Simulation of what we see when

The human visual system is a foveating system

we fixate with cone vision.

Credit: Stuart Anstis, UCSD

Cone distribution and photoreceptor mosaics

Webvision

Andrew Stockman


Arrangement of visual pigment moleculesThe molecule consists of protein, opsin, forming 7 transmembrane α-helices, surrounding the chromophore, retinal, the aldehyde of Vitamin A

Human foveal cones each contain about 1010 visual pigment molecules

Chromophore (chromo- color, + -phore, producer)Light-catching portion of any molecule

11-cis retinal. The molecule is twisted at the 11th carbon.

all-trans retinal Side chains omitted for simplicity.

PhototransductionEnergy of absorbed photon is converted

(transduced) to an electrical neural signal, the receptor potential.

Phototransduction• Activation• Range extension

D i i

Inspired by:h ik b ( )

• Deactivation

Pugh, Nikonov, & Lamb (1999). Current Opinion on Neurobiology, 9, 410-418.Burns & Arshavsky (2005). Neuron, 48, 387-401.

Andrew Stockman


Rhodopsin (R)G-protein

coupled receptor

Main molecular players in the cascade…

p p

Ch h

PDE6Phosphodiesterase,

effector molecule, withα, β and two γ subunits

Transducin (G)G-protein, trimer with α, β and γ subunits

Chromophore11-cis-retinal

In the Dark…

In the Dark

Intracellular cGMP concentration highCNG channels open

CNG = Cyclic Nucleotide Gated channel

Activation steps

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Activation steps

A photon is absorbed resulting in activated R*

Activation steps

Activated transducin, G*α, binds to and activates PDE6 by displacing the γ inhibitory subunit to

produce G*α-PDE6*.R* catalyses the exchange of GDP for GTP on the G-protein,

producing the activated subunit G*α, which dissociates

Activation steps

G*α-PDE6* produces a local drop in cytoplasmic cGMP

The drop in cGMP leads to closure of the CNG channels, which blocks the entry of Na+ and Ca2+ ions into the outer

segment, causing the outer segment to hyperpolarize.

Amplification

The absorption of a single photon is sufficient to change the membrane conductance. How?

A single R* catalyses the activation of c. 500 transducin molecules. Each G*α can stimulate one PDE6*, which in turn can break down 103,molecules of cGMP per second. Thus, a single R* can cause the hydrolysis of >105 molecules of cGMP per second!

Andrew Stockman


R i dRange extension andlight adaptation Why is light adaptation or

sensitivity regulation important?

Because the visual system must maintain itself within a useful operating range over the roughly 1012 change in illumination: from absolute rod threshold to levels at which photoreceptor damage can occur.

Photopic retinal illuminance -4 3 -2 4 -0 5 1 1 2 7 4 5 6 5 8 5

Typical ambient light levels

Indoorlighting

Starlight

Moonlight Sunlight

(log phot td)

Scotopic retinal illuminance(log scot td)

4.3

-3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9

2.4 0.5 1.1 2.7 4.5 6.5 8.5

Absolute rodthreshold

Conethreshold

Rod saturationbegins

Damagepossible

Visual function

It must do so despite the fact that that a typical postreceptoral neuron can operate over a range of only c. 102.

Photopic retinal illuminance -4 3 -2 4 -0 5 1 1 2 7 4 5 6 5 8 5


Indoorlighting

Starlight

Moonlight Sunlight

(log phot td)


4.3

-3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9

2.4 0.5 1.1 2.7 4.5 6.5 8.5


Conethreshold


Damagepossible

Visual function

Andrew Stockman


Photopic retinal illuminance(l h t td)

-4.3 -2.4 -0.5 1.1 2.7 4.5 6.5 8.5


Indoorlighting

Starlight

Moonlight Sunlight

(log phot td)


-3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9

PHOTOPIC


Conethreshold


Damagepossible

SCOTOPICVisual function

MESOPIC

Scotopic levels(below cone threshold)

where rod vision functions alone.A range of c. 103

Photopic levels(above rod saturation)

where cone vision functions alone.A range of > 106

Mesopic levelswhere rod and

cone vision function together.A range of c. 103

Adaptation and sensitivity…A single R* catalyses the activation of c. 500 transducin molecules Each G*α can stimulatetransducin molecules. Each G α can stimulate one PDE6*, which in turn can break down 103

molecules of cGMP per second. Thus, a single R* can cause the hydrolysis of >105 molecules of cGMP per second!

h hi h i i i ill k hBut such high sensitivity will make the system saturate at high light levels?

System must ADAPT to changes in light level

Range extension (1)

Reduction in [Ca2+] causes Calmodulin (CaM) to dissociate from the CNG channels, lowering their K1/2of the channels for cGMP

Need less cGMP to open the CNG channels

Range extension (2)

Reduction in [Ca2+] causes Calmodulin (CaM) to dissociate from the CNG channels, lowering their K1/2of the channels for cGMP

Reduction in [Ca2+] causes dissociation of Ca2+ from

GCAP allowing it to bind toGCAP, allowing it to bind to GC increasing the rate of

resynthesis of cGMP

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Increase in concentration of G*α-PDE6* in light speeds up rate of reaction 2 and speeds up the visual response

Speeding up the visual response

How does speeding up the visual response help light adaptation?

Reduces the integration time of the systemsystem.

Deactivation

Deactivation steps

Rec-2Ca2+ forms a complex with RK, blocking its activity.

When [Ca2+] drops, Ca2+

dissociates and Rec goes into solution.

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Deactivation steps

Free RK multiply phosphorylates R*

Deactivation steps

Arrestin (Arr) quenches the phosphorylated R*

Deactivation steps

(2)

RGS9 deactivates the activated G*α-PDE6* complex.

Deactivation steps

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Second run through…Phototransduction cascade

activation stages

Activation steps of the phototransduction cascade

Credit: Pugh, Nikonov & Lamb

A photon is absorbed resulting in activated R*



R* catalyses the exchange of GDP for GTP on the G-protein, producing the activated transducin, subunit G*α

(Gα-GTP).

Andrew Stockman




Activated transducin, G*α, in turn, binds to and activates phospho-

diesterase (PDE6) by displacing γinhibitory subunits to produce PDE6*.



PDE6* (G*α-E*) activity produces a local drop in cytoplasmic cG (cGMP)


Credit: Pugh, Nikonov & LambA drop in cGMP leads to closure of cGMP gated

channels, blocking the entry of Na+ and Ca2+ into the outer segment. The ion exchanger continues to function lowering [Ca2+] in the outersegment

Phototransduction cascade inactivation steps

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Inactivation steps of the phototransduction cascade


Removal of Ca2+ activates guanylate cyclase activating protein, GCAP. Activated GCAP binds to guanylate cyclase, stimulating production of cG

Ca2+ feedback


In dark [Ca2+], most of recoverin (Rec) is in the calcium bound form at the membrane; Rec-2Ca forms a complex bond with rhodopsin kinase (RK) blocking its activity.

Ca2+ feedback

When [Ca2+] drops Ca dissociates from Rec which


When [Ca ] drops, Ca dissociates from Rec, which moves into solution. Free RK rapidly increases, increasing its interaction with R*, and leading to its rapid phosphorylation.

Ca2+ feedback

Arrestin (Arr) then binds quenching the activity of R*


Arrestin (Arr) then binds quenching the activity of R .

Ca2+ feedback

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G*α-E* is inactivated when terminal phosphate of its bound GTP is hydrolyzed. This is activated when the

RGS9-Gβ5 protein binds.

Summary of molecularadaptation mechanisms

We’ll come back to these in the Sensitivity Regulation lecture

Mechanisms that shorten the visual integration time

[G*α-PDE6*] dependent Increased rate of hydrolysis of cGMP to GMP

[Ca2+] dependent activity of Rec.

Photopigment bleaching (less

Mechanisms that decrease sensitivity

Photopigment bleaching (less photopigment available at high

light levels)

Reduction in the number of open CNG-gated channels

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Mechanisms that increase sensitivity (range extension)

[Ca2+] dependent restoration of cGMP by GC

[Ca2+] increase in CNG channels sensitivity to cGMP

Phototransduction – cones versus rods

Visual pigment, transducin, arrestin

Cones have different isoforms of:

Cones versus rods

PDE6, cGMP channel, and recoverin.

Quantitative differences. In cones:(i) R* forms 4 times faster than for rods - faster onset of light response.

(ii) R* decays 10-50 times faster (lower amplification factor).

(iii) GTPase activating protein (RGS-Gβ5) expressed at much higher levels -(iii) GTPase activating protein (RGS Gβ5) expressed at much higher levels shorter G*α (activated transducin) lifetime - faster recovery.

(iv) Clearance of Ca2+ from cone outer segments is several times faster than for rods.

(v) cGMP channels in cones are twice as permeable to Ca2+ than in rods.

Cones versus rods

Cones are 25 - 100 times less sensitive to single photonsphotons.

They catch fewer photons (less visual pigment).

They respond with faster kinetics (isoforms of transduction cascade).

They have a much greater ability to adapt to y g y pbackground light.

They do not saturate at normal environmental light levels.

Andrew Stockman


Transduction and univariance

Chromophore

How can a process initiatedHow can a process initiated like this encode wavelength

and intensity?

Can it?

Vision at the photoreceptor stage isVision at the photoreceptor stage is relatively simple because the output

of each photoreceptor is:

UNIVARIANTUNIVARIANT

What is univariance?

Probability of photon absorption depends upon wavelengthupon wavelengthBut the response is independent of photon wavelength --

Response varies in ‘one way’—more or less photons are absorbed.A change in output could be caused by a h i i i h i lchange in intensity or a change in colour.

Each photoreceptor is therefore ‘colour blind’, being unable to distinguish between colour and intensity changes.

Andrew Stockman


Univariance in suctionelectrode recordings

Univariance

If a cone is n times less sensitive to light A than to light B, then if A is set to be n times brighter than B, the two lights will appear

identical whatever their wavelengthsidentical whatever their wavelengths.

If we had only one photoreceptor type in our eyes,

what colours would we see?

If we had only one photoreceptor, we would be colour-blind…

Examples: night vision, blue cone monochromats

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So, how do we see colours?

y

0

Four human photoreceptors have different spectral sensitivities

λmax (nm, corneal, quantal)441 500 541 566

Note logarithmic

0 qua

ntal

sen

sitiv

ity

-3

-2

-1 scale

LRods

Wavelength (nm)400 450 500 550 600 650 700

Log 1

-5

-4

MS

Photopigments and spectral tuning

Photopigment molecule (cone)From Sharpe, Stockman, Jägle & Nathans, 1999

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The spectral sensitivity of the photopigment depends on the energy required to flip the chromophore from it 11-cisform to its all-trans form.

But the same chromophore is used in all four human photo-pigments.

The energy is modified by changes in the amino acids in th di i

Photopigment molecule (cone)From Sharpe, Stockman, Jägle & Nathans, 1999

the surrounding opsinmolecule.

Opsin differences

From Sharpe, Stockman, Jägle & Nathans, 1999

15 amino acid differences -96% identical

C-1

C-2C-3

360

80

160 250260 330

340

350

SS

SSS

V

V

Cytoplasmic side

MWS/LWS cone opsin tuning Phosphorylation sites

Transducin, arrestin activation regions

C-terminal

I

EC

C

K

60

70

80

90

100

110 130

140

150

170

180

190220

230

240270

280

290

300

310

320

340

SS

Membrane

ExtracellularE-1

E-2

E-3C

1

10

20

30

40

50

120

200

210

300

S

Extracellular side

Retinal binding site (Lys312)Schiff base counterion (Glu129)Chloride binding pocket: Glu-197His+ (& Gln200Lys+)

KE

N-terminal

Spectral tuning sites180, 277, 285

POLA

R

LWS all with OH groupP

-PO

LAR

OH group

MWSN

ON

- MWS

164/180 261/277 269/285Rods and S cones have 348 amino acids whereas L and M cones

have 364 amino acids – 16 amino acid difference at the N terminal

Andrew Stockman


MWS LWS

Tuning Site

164/180 alanine serine OH- ~ 5 nm

261/277 phenylalanine tyrosine OH-

~ 25 nm

269/285 alanine threonine OH-

25 nm

y

0

Four human photoreceptors have different spectral sensitivities

λmax (nm, corneal, quantal)441 500 541 566

Note logarithmic

0 qua

ntal

sen

sitiv

ity

-3

-2

-1 scale

LRods

Wavelength (nm)400 450 500 550 600 650 700

Log 1

-5

-4

MS

No colour… With three cone types, we see colour…

Andrew Stockman


People with normal colour vision have three univariant cones with different spectral sensitivities…p

Their colour vision is therefore three dimensional or:

TRICHROMATICTRICHROMATIC

Additive colour mixing

Red+Green+Blue

y

0

Four photoreceptors have different spectral sensitivities441 500 541 566

0 qua

ntal

sen

sitiv

ity

-3

-2

-1

L-cones

Wavelength (nm)

400 450 500 550 600 650 700

Log 1

-5

-4

S-cones M-conesRods

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