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Lecture # 14

Lecture # 14. Vertebrate phototransduction 3 /14/13. Midterm. Thanks to student questioners Sonia – Silurians Sarah – Lake Cerise Jessica – Arthur’s glasses Brian – lemur vision Still grading Midterm 20% HW 50% of grade Wide distribution Work together after break.

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Lecture # 14

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  1. Lecture # 14 Vertebrate phototransduction 3/14/13

  2. Midterm • Thanks to student questioners • Sonia – Silurians Sarah – Lake Cerise • Jessica – Arthur’s glasses Brian – lemur vision • Still grading • Midterm 20% HW 50% of grade • Wide distribution • Work together after break

  3. Wiki – Animal vision project • Think about an animal whose visual system you want to learn more about • Tuesday after break we will sign up for animals and learn about creating wiki pages

  4. Today • How signal transduction works in photoreceptors • Light in = neural signal out • Why photoreceptors are weird • How rods and cones differ • Let us count the ways • Evolution of two pathways

  5. Phototransduction • Transduction • “the conversion of a signal from one form to another” • Photo - signal comes from light • Transduce – neural signal goes out

  6. Typical neuron

  7. Ion pump creates a concentration gradient across cell membrane Na+ 150 mM 120 mM 5mM 15 mM 10 mM 140 mM Na+ Cl- Cl- Na/K ATPase K+ K+ Outside cell Inside cell

  8. Leaky K+ channels lets out K+ which makes inside of cell negative Na+ 150 mM 120 mM 5 mM - - - - - - - - - - 15 mM 10 mM 140 mM Na+ Cl- Cl- Na/K ATPase K+ K+ Outside cell Inside cell

  9. Na+ channel opens and sodium goes into cell : down concentration and potential gradient Na+ 150 mM 120 mM 5 mM - - - - - - - - - - 15 mM 10 mM 140 mM Na+ Cl- Cl- Na/K ATPase K+ K+ Outside cell Inside cell

  10. Photoreceptor parts • Outer segment • Lots of membrane • Where light gets detected • Inner segment • Mitochondria to power cell • Nucleus - DNA • Synapse • Sends signal to next neuron

  11. Rods: Current flows in dark • Ion pump moves ions across membrane • cGMP gated channels are open in dark • Na+flows back in • Channels open when signal is NOT present • Circulating “dark” current

  12. Under dark conditions • Channels are open (Na+ flows in) • Circulating “dark” current • Membrane potential is -35 mV • Partial depolarization results in glutamate being constantly released Glutamate release

  13. Electrophysiology Measure membrane current in rod photoreceptor Current decreases with light = channels close

  14. Light closes channels • This prevents Na+ from flowing in • But K+ and Ca+2 are still being sent out (exchanger) • Inside of cell gets more negative • Hyperpolarizes • Circulating current decreases

  15. When light is absorbed • Channels close • Hyperpolarization - membrane potential gets more negative • Glutamate decreases • Glutamate release is variable: photoreceptor is continuously responding. • Glutamate change signals next cells L I G H T Less glutamate released

  16. When light is absorbed • Channels close • Hyperpolarization - membrane potential gets more negative • Glutamate decreases • Glutamate release is variable: photoreceptor is continuously responding. • Glutamate change signals next cells L I G H T Less glutamate released

  17. Rod structure – outer cell membrane with stack of discs inside

  18. Phototransduction • How does photon signal get from visual pigment to synapse? • Signal to close ion channels • Hyperpolarization decreases Ca level • Lower calcium causes less glutamate release

  19. The players • Visual pigment • Opsin protein surrounds 11-cis retinal • Combination absorb light

  20. The players • G protein • Three subunits • α binds GDP / GTP • βγ binds inactive α • Activates effector • For vision it is α

  21. Phosphodiesterase - effector • Two catalytic subunits α and β • Can convert cGMP to GMP • Two inhibitory subunits γ • Gα* inhibits the gamma subunits and turns on catalysis α β γ γ

  22. cGMP gated ion channel • Cooperatively binds 4 cGMP • When cGMP is bound, channel is open cG cG cG cG

  23. G protein pathway in rod disc Rhodopsin G protein R + hvgR* Rhodopsin absorbs photon g excited R* + GαβγgR* + Gα*-GTP + Gβγ Rhodopsin activates G protein

  24. G protein pathway in rod disc Rhodopsin G proteinE,phosphodiesterase R + hvgR* Rhodopsin absorbs photon g excited R* + GαβγgR* + Gα*-GTP + Gβγ Rhodopsin activates G protein Gα* + E gE* G protein activates phosphodiesterase, E actually inhibits the inhibitory γ subunit E* + cGMPg GMPPhosphodiesesterase causes cGMP decrease

  25. G protein pathway in rods Channels are gated by cGMP. As cGMP decreases, it dissociates from open channel, closing it. This prevents Na+ from entering cell. Ca2+ and K+ are still being sent out of the cell through the exchanger, so charge inside cell gets more negative.

  26. G protein pathway in rods Note the exchanger It pumps Ca and K out and Na in Always working

  27. Phototransduction video

  28. G protein pathway in rods All the players work together to close channel and cause hyperpolarization

  29. Phototransduction • Relative proportion of proteins • Rhodopsin - 1000 • Transducin - 100 • PDE - 4

  30. Gain in this signal transduction? Note: R* stays activated after it has activated G protein. One R* can activate up to 700 G* which each activate 1 E*. One E* can hydrolyze about 8cGMP So one photon leads to hydrolysis of 5600 cGMP

  31. When light is absorbed • Channels close • Hyperpolarization - membrane potential gets more negative • Glutamate decreases • Glutamate release is variable: photoreceptor is continuously responding. • Glutamate signals next cells L I G H T Less glutamate released

  32. Circulating current • What happens if all channels close?? • Current goes to zero as light level increases

  33. How are they the same? • Same – Gproteins sometimes; same Ca/ Na/K ions Graded response to change - • Depolarization = neurotransmitter output • Hyperpolarization = neurotranmitter decrease

  34. How are rods weird / different from other sensory neurons? • Signal = hyperpolarization • Signal = channels closing • Signal = less neurotranmitter

  35. Turning off excitation-recovery #4 reopen channels #3 make cGMP #1 shut off R* #2 shut off E*

  36. R* shutoff Note: Only after arrestin binds does all trans retinal dissociate!!

  37. E* shutoff RGS = regulator of G protein signaling

  38. Regenerate cGMP by guanylate cyclase

  39. All kinds of feedback to make recovery faster if high light levels : Ca2+signalling #3 make cGMP #1 shut off R* #2 shut off E*

  40. Electrophysiology Measure membrane current in rod photoreceptor Current decreases with light = channels close

  41. Circulating current decreases • As flash more light, channels close and current drops • Then current recovers as channels open again

  42. How many ways do rods and cones differ?

  43. Rods and cones differ 1. Morphology of outer segment

  44. Disks are distinct in rods • Special proteins in rim help disks to form • Peripherin • Rom-1 • ABCR/Rim • moves retinal across membrane

  45. Rods and cones differ2. Spectral sensitivity Rod 498 nm (11) Green 534 nm (11) Blue 420 nm (3) Red 564 nm (19) Bowmaker and Dartnall 1980

  46. Rods and cones differ 3. Location and number

  47. Rods and cones differ #4 Electrophysiology Suction pipette measures membrane current In response to light: current decreases because channels close

  48. Rods and cones differ in how respond to light • Rods • High sensitivity • Saturate • Slow • Cones • Low sensitivity • Big dynamic range • Fast

  49. Rods and cones differ #4. Electrophysiology Circulating current decreases NOTE – decrease in current is up on y axis

  50. Relative sensitivity Rods can detect single photons cones Absolute threshold Rod saturation rods 10-4 10-3 10-2 0.1 1 10 102 103 104 105 106 Photons / sec

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