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IoO MSc Phototransduction and photopigments - Microsoft PowerPoint ... IoO MSc Phototransduction and photopigments - Microsoft PowerPoint ... Presentation Transcript

  • Andrew Stockman Phototransduction and photopigments Background MSc BIOLOGY OF VISION Introduction to Visual Neuroscience Andrew Stockman The retina is carpeted with light- Light 400 - 700 nm is important for vision sensitive rods and cones An inverted image is formed on the retinaPhototransduction & Photopigments 1
  • Andrew Stockman Rods and Human photoreceptors cones Rods Cones Achromatic Daytime, achromatic night vision g and chromatic vision 1 type 3 types Rod Long-wavelength- sensitive (L) or “red” cone Middle-wavelength- sensitive (M) or “green” cone Short-wavelength- sensitive (S) or “blue” cone Webvision Rod and cone distribution Rod density peaks at about 20 deg eccentricity At night, you have to look 0.3 mm of eccentricity is away from things to see about 1 deg of visual angle them in more detailPhototransduction & Photopigments 2
  • Andrew Stockman Original photograph The human visual system During the day, you have to look at Cones peak at the centre is a foveating system things directly to see them in detail of vision at 0 deg Simulation of what we see when we fixate with cone vision. Credit: Stuart Anstis, UCSD Cone distribution and photoreceptor mosaics WebvisionPhototransduction & Photopigments 3
  • Andrew Stockman 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 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. Human foveal cones each contain about 1010 visual pigment molecules Phototransduction • Activation • Range extension Phototransduction • D Deactivation i i Energy of absorbed photon is converted (transduced) to an electrical neural signal, the receptor potential. Inspired by: Pugh, Nikonov, & Lamb (1999). h ik b( ) Current Opinion on Neurobiology, 9, 410-418. Burns & Arshavsky (2005). Neuron, 48, 387-401.Phototransduction & Photopigments 4
  • Andrew Stockman Main molecular players in the cascade… Rhodopsin (R) G-protein coupled receptor p p In the Dark… Chromophore Ch h 11-cis-retinal Transducin (G) PDE6 Phosphodiesterase, G-protein, trimer with effector molecule, with α, β and γ subunits α, β and two γ subunits In the Dark Activation steps Intracellular cGMP concentration high CNG channels open CNG = Cyclic Nucleotide Gated channelPhototransduction & Photopigments 5
  • Andrew Stockman Activation steps Activation steps A photon is absorbed resulting in activated R* Activated transducin, G*α, binds to and activates the exchange of GDP γ GTP on subunit to R* catalysesPDE6 by displacing theforinhibitory the G-protein, produce G*α-PDE6*. producing the activated subunit G*α, which dissociates Activation steps 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! The drop in cGMP leads to closure of the CNG channels, which blocks the entry of Na+ andlocal ions into the outer G*α-PDE6* produces a Ca2+ drop in cytoplasmic cGMP segment, causing the outer segment to hyperpolarize.Phototransduction & Photopigments 6
  • Andrew Stockman Range extension and R i d light adaptation Why is light adaptation or sensitivity regulation important? Because the visual system must maintain itself within a It must do so despite the fact that that a typical useful operating range over the roughly 1012 change in postreceptoral neuron can operate over a range of illumination: from absolute rod threshold to levels at only c. 102. which photoreceptor damage can occur. Moonlight Sunlight Moonlight Sunlight Typical ambient light levels Typical ambient light levels Starlight Indoor Starlight Indoor lighting lighting Photopic retinal illuminance Photopic retinal illuminance -4 3 4.3 -2 4 2.4 -0 5 0.5 1.1 11 2.7 27 4.5 45 6.5 65 8.5 85 -4 3 4.3 -2 4 2.4 -0 5 0.5 1.1 11 2.7 27 4.5 45 6.5 65 8.5 85 (log phot td) (log phot td) Scotopic retinal illuminance -3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9 Scotopic retinal illuminance -3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9 (log scot td) (log scot td) Visual function Visual function Absolute rod Cone Rod saturation Damage Absolute rod Cone Rod saturation Damage threshold threshold begins possible threshold threshold begins possiblePhototransduction & Photopigments 7
  • Andrew Stockman Typical ambient light levels Moonlight Sunlight Adaptation and sensitivity… Starlight Indoor lighting A single R* catalyses the activation of c. 500 Photopic retinal illuminance (log h t (l phot td) -4.3 -2.4 -0.5 1.1 2.7 4.5 6.5 8.5 transducin molecules. Each G α can stimulate molecules G*α Scotopic retinal illuminance -3.9 -2.0 -0.1 1.5 3.1 4.9 6.9 8.9 one PDE6*, which in turn can break down 103 (log scot td) SCOTOPIC MESOPIC PHOTOPIC molecules of cGMP per second. Thus, a single R* Visual function can cause the hydrolysis of >105 molecules of cGMP per second! Absolute rod Cone Rod saturation Damage threshold threshold begins possible But such hi h sensitivity will make the h high ii i ill k h Scotopic levels Mesopic levels Photopic levels system saturate at high light levels? (below cone threshold) where rod and (above rod saturation) where rod vision cone vision where cone vision functions alone. function together. functions alone. System must ADAPT to changes in light level A range of c. 103 A range of c. 103 A range of > 106 Range extension (1) Range extension (2) Reduction in [Ca2+] causes Reduction in [Ca2+] causes Calmodulin (CaM) to Calmodulin (CaM) to dissociate from the CNG dissociate from the CNG channels, lowering their K1/2 channels, lowering their K1/2 of the channels for cGMP of the channels for cGMP Need less cGMP to open the CNG channels Reduction in [Ca2+] causes dissociation of Ca2+ from GCAP, GCAP allowing it to bind to GC increasing the rate of resynthesis of cGMPPhototransduction & Photopigments 8
  • Andrew Stockman Speeding up the visual response How does speeding up the visual response Increase in concentration of G*α-PDE6* in light speeds up rate of reaction 2 and speeds up the visual response help light adaptation? Reduces the integration time of the system. system Deactivation steps Deactivation Rec-2Ca2+ forms a complex with RK, blocking its activity. When [Ca2+] drops, Ca2+ dissociates and Rec goes into solution.Phototransduction & Photopigments 9
  • Andrew Stockman Deactivation steps Deactivation steps Free RK multiply Arrestin (Arr) quenches phosphorylates R* the phosphorylated R* Deactivation steps Deactivation steps (2) RGS9 deactivates the activated G*α-PDE6* complex.Phototransduction & Photopigments 10
  • Andrew Stockman Phototransduction cascade Second run through… activation stages Activation steps of the phototransduction cascade Activation steps of the phototransduction cascade R* catalyses the exchange of GDP A photon is absorbed for GTP on the G-protein, producing resulting in activated R* the activated transducin, subunit G*α Credit: Pugh, Nikonov & Lamb Credit: Pugh, Nikonov & Lamb (Gα-GTP).Phototransduction & Photopigments 11
  • Andrew Stockman Activation steps of the phototransduction cascade Activation steps of the phototransduction cascade Activated transducin, G*α, in turn, binds to and activates phospho- PDE6* (G*α-E*) activity produces a diesterase (PDE6) by displacing γ local drop in cytoplasmic cG (cGMP) Credit: Pugh, Nikonov & Lamb Credit: Pugh, Nikonov & Lamb inhibitory subunits to produce PDE6*. Activation steps of the phototransduction cascade Phototransduction cascade inactivation steps A drop in cGMP leads to closure of cGMP gated Credit: Pugh, Nikonov & Lamb channels, blocking the entry of Na+ and Ca2+ into the outer segment. The ion exchanger continues to function lowering [Ca2+] in the outersegmentPhototransduction & Photopigments 12
  • Andrew Stockman Inactivation steps of the phototransduction cascade In dark [Ca2+], most of recoverin (Rec) is in the calcium Removal of Ca2+ activates guanylate bound form at the membrane; Rec-2Ca forms a complex cyclase activating protein, GCAP. Ca2+ feedback bond with rhodopsin kinase (RK) blocking its activity. Activated GCAP binds to guanylate cyclase, stimulating production of cG Credit: Pugh, Nikonov & Lamb Ca2+ feedback Credit: Pugh, Nikonov & Lamb When [Ca2+] drops, Ca dissociates from Rec, which drops Rec Arrestin (Arr) then binds quenching the activity of R . R* moves into solution. Free RK rapidly increases, increasing its interaction with R*, and leading to its rapid Ca2+ feedback phosphorylation. Ca2+ feedback Credit: Pugh, Nikonov & Lamb Credit: Pugh, Nikonov & LambPhototransduction & Photopigments 13
  • Andrew Stockman Summary of molecular adaptation mechanisms G*α-E* is inactivated when terminal phosphate of its bound GTP is hydrolyzed. This is activated when the RGS9-Gβ5 protein binds. We’ll come back to these in the Sensitivity Regulation lecture Credit: Pugh, Nikonov & Lamb Mechanisms that shorten the visual integration time Mechanisms that decrease sensitivity Photopigment bleaching (less [G*α-PDE6*] dependent Increased photopigment available at high rate of hydrolysis of cGMP to GMP light levels) [Ca2+] dependent activity of Rec. Reduction in the number of open CNG-gated channelsPhototransduction & Photopigments 14
  • Andrew Stockman Mechanisms that increase sensitivity (range extension) [Ca2+] dependent restoration of cGMP by GC Phototransduction – cones versus rods [Ca2+] increase in CNG channels sensitivity to cGMP Cones versus rods Cones versus rods Cones have different isoforms of: Cones are 25 - 100 times less sensitive to single Visual pigment, transducin, arrestin photons. photons PDE6, cGMP channel, and recoverin. They catch fewer photons (less visual pigment). Quantitative differences. In cones: (i) R* forms 4 times faster than for rods - faster onset of light response. They respond with faster kinetics (isoforms of transduction cascade). (ii) R* decays 10-50 times faster (lower amplification factor). (iii) GTPase activating protein (RGS-Gβ5) expressed at much higher levels - (RGS Gβ5) They have a much g y greater ability to adapt to y p shorter G*α (activated transducin) lifetime - faster recovery. background light. (iv) Clearance of Ca2+ from cone outer segments is several times faster than for rods. They do not saturate at normal environmental light levels. (v) cGMP channels in cones are twice as permeable to Ca2+ than in rods.Phototransduction & Photopigments 15
  • Andrew Stockman Chromophore How can a process initiated like this encode wavelength Transduction and univariance and intensity? Can it? What is univariance? Probability of photon absorption depends Vision at the photoreceptor stage is upon wavelength But the response is independent of photon relatively simple because the output wavelength -- of each photoreceptor is: Response varies in ‘one way’—more or less photons are absorbed. A change in output could be caused by a UNIVARIANT change in intensity or a change in colour. h i i i h i l Each photoreceptor is therefore ‘colour blind’, being unable to distinguish between colour and intensity changes.Phototransduction & Photopigments 16
  • Andrew Stockman Univariance If a cone is n times less sensitive to light A Univariance in suction electrode recordings than to light B, then if A is set to be n times brighter than B, the two lights will appear identical whatever their wavelengths. wavelengths If we had only one photoreceptor, we would be colour-blind… If we had only one photoreceptor type in our eyes, what colours would we see? Examples: night vision, blue cone monochromatsPhototransduction & Photopigments 17
  • Andrew Stockman Four human photoreceptors have different spectral sensitivities 441 500 541 566 λmax (nm, corneal, quantal) 0 Note logarithmic Log10 quantal sensitivity y scale -1 So, how do we see colours? -2 -3 Rods L -4 S -5 M 400 450 500 550 600 650 700 Wavelength (nm) Photopigments and spectral tuning Photopigment molecule (cone) From Sharpe, Stockman, Jägle & Nathans, 1999Phototransduction & Photopigments 18
  • Andrew Stockman The spectral sensitivity of the Opsin differences 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 the th surrounding opsin di i molecule. 15 amino acid differences - 96% identical Photopigment molecule (cone) From Sharpe, Stockman, Jägle & Nathans, 1999 From Sharpe, Stockman, Jägle & Nathans, 1999 MWS/LWS cone opsin tuning C-terminal Phosphorylation S sites V S 360 Transducin, arrestin 350 S SSV activation regions C-3 C-2 POLAR Cytoplasmic C-1 160 250 260 330 LWS side all with 340 OH group P 80 150 90 240 170 270 320 70 140 Membrane I 180 280 100 230 K 310 -POLAR 130 110 60 E C 220 S 190 290 Extracellular S side 50 C 200 300 NON- MWS 120 E-1 E-3 Spectral tuning sites 210 20 180, 277, 285 S 40 E-2 K Retinal binding site (Lys312) E Schiff base counterion (Glu129) 10 164/180 261/277 269/285 30 Chloride binding pocket: 1 Rods and S cones have 348 amino acids whereas L and M cones Glu-197His+ (& Gln200Lys+) N-terminal have 364 amino acids – 16 amino acid difference at the N terminalPhototransduction & Photopigments 19
  • Andrew Stockman Four human photoreceptors have different spectral sensitivities MWS LWS 441 500 541 566 λmax (nm, corneal, quantal) Tuning Site 164/180 alanine serine OH- ~ 5 nm 0 Note logarithmic Log10 quantal sensitivity y scale -1 261/277 phenylalanine tyrosine OH- -2 -3 Rods ~ 25 nm L -4 S 269/285 alanine threonine OH- -5 M 400 450 500 550 600 650 700 Wavelength (nm) No colour… With three cone types, we see colour…Phototransduction & Photopigments 20
  • Andrew Stockman Additive colour mixing People with normal colour vision have three univariant cones with different spectral sensitivities… p Their colour vision is therefore three dimensional or: TRICHROMATIC Red+Green+Blue Four photoreceptors have different spectral sensitivities 441 500 541 566 0 Log10 quantal sensitivity y -1 -2 -3 L-cones -4 -5 S-cones Rods M-cones 400 450 500 550 600 650 700 Wavelength (nm)Phototransduction & Photopigments 21