Andrew Stockman
Phototransduction & Photopigments 1
Phototransduction and photopigments
Andrew Stockman
MSc BIOLOGY OF VIS...
Andrew Stockman
Phototransduction & Photopigments 2
Rods and
cones
Webvision
Human photoreceptors
Rods
Achromatic
night vi...
Andrew Stockman
Phototransduction & Photopigments 3
During the day, you have to look at
things directly to see them in det...
Andrew Stockman
Phototransduction & Photopigments 4
Arrangement of visual pigment molecules
The molecule consists of prote...
Andrew Stockman
Phototransduction & Photopigments 5
Rhodopsin (R)
G-protein
coupled receptor
Main molecular players in the...
Andrew Stockman
Phototransduction & Photopigments 6
Activation steps
A photon is absorbed
resulting in activated R*
Activa...
Andrew Stockman
Phototransduction & Photopigments 7
R i dRange extension and
light adaptation Why is light adaptation or
s...
Andrew Stockman
Phototransduction & Photopigments 8
Photopic retinal illuminance
(l h t td)
-4.3 -2.4 -0.5 1.1 2.7 4.5 6.5...
Andrew Stockman
Phototransduction & Photopigments 9
Increase in concentration of G*α-PDE6* in light speeds up
rate of reac...
Andrew Stockman
Phototransduction & Photopigments 10
Deactivation steps
Free RK multiply
phosphorylates R*
Deactivation st...
Andrew Stockman
Phototransduction & Photopigments 11
Second run through…
Phototransduction cascade
activation stages
Activ...
Andrew Stockman
Phototransduction & Photopigments 12
Activation steps of the phototransduction cascade
Credit: Pugh, Nikon...
Andrew Stockman
Phototransduction & Photopigments 13
Inactivation steps of the phototransduction cascade
Credit: Pugh, Nik...
Andrew Stockman
Phototransduction & Photopigments 14
Credit: Pugh, Nikonov & Lamb
G*α-E* is inactivated when terminal phos...
Andrew Stockman
Phototransduction & Photopigments 15
Mechanisms that increase sensitivity (range extension)
[Ca2+] depende...
Andrew Stockman
Phototransduction & Photopigments 16
Transduction and univariance
Chromophore
How can a process initiatedH...
Andrew Stockman
Phototransduction & Photopigments 17
Univariance in suction
electrode recordings
Univariance
If a cone is ...
Andrew Stockman
Phototransduction & Photopigments 18
So, how do we see colours?
y
0
Four human photoreceptors have differe...
Andrew Stockman
Phototransduction & Photopigments 19
The spectral sensitivity of the
photopigment depends on the
energy re...
Andrew Stockman
Phototransduction & Photopigments 20
MWS LWS
Tuning Site
164/180 alanine serine OH- ~ 5 nm
261/277 phenyla...
Andrew Stockman
Phototransduction & Photopigments 21
People with normal colour vision have three univariant
cones with dif...
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IoO MSc Phototransduction and photopigments - Microsoft PowerPoint ...

  1. 1. Andrew Stockman Phototransduction & Photopigments 1 Phototransduction and photopigments Andrew Stockman MSc BIOLOGY OF VISION Introduction to Visual Neuroscience Background Light 400 - 700 nm is important for vision The retina is carpeted with light- sensitive rods and cones An inverted image is formed on the retina
  2. 2. Andrew Stockman Phototransduction & Photopigments 2 Rods and cones Webvision Human photoreceptors Rods Achromatic night vision Cones Daytime, 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
  3. 3. Andrew Stockman Phototransduction & Photopigments 3 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
  4. 4. Andrew Stockman Phototransduction & Photopigments 4 Arrangement of visual pigment molecules The 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. Phototransduction Energy 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.
  5. 5. Andrew Stockman Phototransduction & Photopigments 5 Rhodopsin (R) G-protein coupled receptor Main molecular players in the cascade… p p Ch h PDE6 Phosphodiesterase, effector molecule, with α, β and two γ subunits Transducin (G) G-protein, trimer with α, β and γ subunits Chromophore 11-cis-retinal In the Dark… In the Dark Intracellular cGMP concentration high CNG channels open CNG = Cyclic Nucleotide Gated channel Activation steps
  6. 6. Andrew Stockman Phototransduction & Photopigments 6 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!
  7. 7. Andrew Stockman Phototransduction & Photopigments 7 R i dRange extension and light 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 Indoor lighting 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 rod threshold Cone threshold Rod saturation begins Damage possible 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 Typical ambient light levels Indoor lighting 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 rod threshold Cone threshold Rod saturation begins Damage possible Visual function
  8. 8. Andrew Stockman Phototransduction & Photopigments 8 Photopic retinal illuminance (l h t td) -4.3 -2.4 -0.5 1.1 2.7 4.5 6.5 8.5 Typical ambient light levels Indoor lighting Starlight Moonlight Sunlight (log phot td) Scotopic retinal illuminance (log scot td) -3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9 PHOTOPIC Absolute rod threshold Cone threshold Rod saturation begins Damage possible SCOTOPIC Visual 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 levels where 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 K1/2 of 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 K1/2 of 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
  9. 9. Andrew Stockman Phototransduction & Photopigments 9 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.
  10. 10. Andrew Stockman Phototransduction & Photopigments 10 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
  11. 11. Andrew Stockman Phototransduction & Photopigments 11 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* Activation steps of the phototransduction cascade Credit: Pugh, Nikonov & Lamb R* catalyses the exchange of GDP for GTP on the G-protein, producing the activated transducin, subunit G*α (Gα-GTP).
  12. 12. Andrew Stockman Phototransduction & Photopigments 12 Activation steps of the phototransduction cascade Credit: Pugh, Nikonov & Lamb Activated transducin, G*α, in turn, binds to and activates phospho- diesterase (PDE6) by displacing γ inhibitory subunits to produce PDE6*. Activation steps of the phototransduction cascade Credit: Pugh, Nikonov & Lamb PDE6* (G*α-E*) activity produces a local drop in cytoplasmic cG (cGMP) Activation steps of the phototransduction cascade Credit: Pugh, Nikonov & Lamb A 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
  13. 13. Andrew Stockman Phototransduction & Photopigments 13 Inactivation steps of the phototransduction cascade Credit: Pugh, Nikonov & Lamb Removal of Ca2+ activates guanylate cyclase activating protein, GCAP. Activated GCAP binds to guanylate cyclase, stimulating production of cG Ca2+ feedback Credit: Pugh, Nikonov & Lamb 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 Credit: Pugh, Nikonov & Lamb 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* Credit: Pugh, Nikonov & Lamb Arrestin (Arr) then binds quenching the activity of R . Ca2+ feedback
  14. 14. Andrew Stockman Phototransduction & Photopigments 14 Credit: Pugh, Nikonov & Lamb 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 molecular adaptation 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
  15. 15. Andrew Stockman Phototransduction & Photopigments 15 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 toy g y p background light. They do not saturate at normal environmental light levels.
  16. 16. Andrew Stockman Phototransduction & Photopigments 16 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 wavelength But 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.
  17. 17. Andrew Stockman Phototransduction & Photopigments 17 Univariance in suction electrode 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
  18. 18. Andrew Stockman Phototransduction & Photopigments 18 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 0quantalsensitivity -3 -2 -1 scale L Rods Wavelength (nm) 400 450 500 550 600 650 700 Log1 -5 -4 M S Photopigments and spectral tuning Photopigment molecule (cone) From Sharpe, Stockman, Jägle & Nathans, 1999
  19. 19. Andrew Stockman Phototransduction & Photopigments 19 The spectral sensitivity of the photopigment depends on the energy required to flip the chromophore from it 11-cis form 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 opsin molecule. Opsin differences From Sharpe, Stockman, Jägle & Nathans, 1999 15 amino acid differences - 96% identical C-1 C-2 C-3 360 80 160 250 260 330 340 350 S S S SS V V Cytoplasmic side MWS/LWS cone opsin tuning Phosphorylation sites Transducin, arrestin activation regions C-terminal I E C C K 60 70 80 90 100 110 130 140 150 170 180 190 220 230 240 270 280 290 300 310 320 340 S S Membrane Extracellular E-1 E-2 E-3 C 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+) K E N-terminal Spectral tuning sites 180, 277, 285 POLAR LWS all with OH group P-POLAR OH group MWSNON- MWS 164/180 261/277 269/285 Rods and S cones have 348 amino acids whereas L and M cones have 364 amino acids – 16 amino acid difference at the N terminal
  20. 20. Andrew Stockman Phototransduction & Photopigments 20 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 0quantalsensitivity -3 -2 -1 scale L Rods Wavelength (nm) 400 450 500 550 600 650 700 Log1 -5 -4 M S No colour… With three cone types, we see colour…
  21. 21. Andrew Stockman Phototransduction & Photopigments 21 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 sensitivities 441 500 541 566 0quantalsensitivity -3 -2 -1 L-cones Wavelength (nm) 400 450 500 550 600 650 700 Log1 -5 -4 S-cones M-conesRods

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