Lecture # 14
description
Transcript of Lecture # 14
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Lecture # 14
Vertebrate phototransduction3/14/13
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Midterm
• Thanks to student questionersSonia – Silurians Sarah – Lake CeriseJessica – Arthur’s glasses Brian – lemur vision
• Still gradingMidterm 20% HW 50% of gradeWide distributionWork together after break
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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
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Today
• How signal transduction works in photoreceptorsLight in = neural signal outWhy photoreceptors are weird
• How rods and cones differLet us count the waysEvolution of two pathways
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Phototransduction
• Transduction “the conversion of a signal from one form to another”
Photo - signal comes from light
Transduce – neural signal goes out
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Typical neuron
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Ion pump creates a concentration gradient across cell membrane
Na/K ATPase
Outside cell Inside cell
Na+
K+
Cl-
15 mM
10 mM
140 mM
Na+
K+
Cl-
150 mM
120 mM
5 mM
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Leaky K+ channels lets out K+ which makes inside of cell negative
Na/K ATPase
Outside cell Inside cell
Na+
K+
Cl-
15 mM
10 mM
140 mM
Na+
K+
Cl-
150 mM
120 mM
5 mM
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Na+ channel opens and sodium goes into cell : down concentration and potential gradient
Na/K ATPase
Outside cell Inside cell
Na+
K+
Cl-
15 mM
10 mM
140 mM
Na+
K+
Cl-
150 mM
120 mM
5 mM
-----
-----
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Photoreceptor parts• Outer segment
Lots of membraneWhere light gets detected
• Inner segmentMitochondria to power cellNucleus - DNA
• SynapseSends signal to next neuron
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Rods: Current flows in dark
• Ion pump moves ions across membrane
• cGMP gated channels are open in darkNa+ flows back in
• Channels open when signal is NOT present
• Circulating “dark” current
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Under dark conditions• Channels are
open (Na+ flows in)
• Circulating “dark” current
• Membrane potential is -35 mV
• Partial depolarization results in glutamate being constantly releasedGlutamate release
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Measure membrane current in rod photoreceptor
Current decreases with light= channels close
Electrophysiology
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Light closes channels
• This prevents Na+ from flowing inBut K+ and Ca+2 are still being sent out (exchanger)
• Inside of cell gets more negativeHyperpolarizes
• Circulating current decreases
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When light is absorbed• Channels close• Hyperpolarization -
membrane potential gets more negative
• Glutamate decreasesGlutamate release is variable: photoreceptor is continuously responding.
• Glutamate change signals next cells
Less glutamate released
LIGHT
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When light is absorbed• Channels close• Hyperpolarization -
membrane potential gets more negative
• Glutamate decreasesGlutamate release is variable: photoreceptor is continuously responding.
• Glutamate change signals next cells
Less glutamate released
LIGHT
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Rod structure – outer cell membrane with stack of discs inside
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Phototransduction
• How does photon signal get from visual pigment to synapse?1. Signal to close ion channels2. Hyperpolarization decreases Ca level3. Lower calcium causes less glutamate release
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The players
• Visual pigment
Opsin protein surrounds 11-cis retinal
Combination absorb light
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The players
• G proteinThree subunitsα binds GDP / GTPβγ binds inactive α
• Activates effectorFor vision it is α
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Phosphodiesterase - effector
• Two catalytic subunits α and βCan convert cGMP to GMP
• Two inhibitory subunits γ
• Gα* inhibits the gamma subunits and turns on catalysis
α β γγ
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cGMP gated ion channel
• Cooperatively binds 4 cGMP
• When cGMP is bound, channel is open
cGcG
cGcG
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G protein pathway in rod disc
R + hv g R* Rhodopsin absorbs photon g excitedR* + Gαβγ g R* + Gα*-GTP + Gβγ Rhodopsin activates G protein
Rhodopsin G protein
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G protein pathway in rod disc
R + hv g R* Rhodopsin absorbs photon g excitedR* + Gαβγ g R* + Gα*-GTP + Gβγ Rhodopsin activates G proteinGα* + E g E* G protein activates phosphodiesterase, E
actually inhibits the inhibitory γ subunitE* + cGMP g GMP Phosphodiesesterase causes cGMP decrease
Rhodopsin G protein E,phosphodiesterase
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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.
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G protein pathway in rods
Note the exchangerIt pumps Ca and K out and Na inAlways working
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Phototransduction video
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G protein pathway in rods
All the players work together to close channel and cause hyperpolarization
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Phototransduction
• Relative proportion of proteins
Rhodopsin - 1000
Transducin - 100
PDE - 4
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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 8 cGMP
So one photon leads to hydrolysis of 5600 cGMP
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When light is absorbed• Channels close• Hyperpolarization -
membrane potential gets more negative
• Glutamate decreasesGlutamate release is variable: photoreceptor is continuously responding.
• Glutamate signals next cells
Less glutamate released
LIGHT
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Circulating current• What happens if all
channels close??• Current goes to zero
as light level increases
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How are they the same?
• Same – Gproteins sometimes; same Ca/ Na/K ions Graded response to change - Depolarization = neurotransmitter outputHyperpolarization = neurotranmitter decrease
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How are rods weird / different from other sensory neurons?
• Signal = hyperpolarization• Signal = channels closing• Signal = less neurotranmitter
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Turning off excitation-recovery
#1 shut off R* #2 shut off E*
#3 make cGMP
#4 reopen channels
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R* shutoff
Note: Only after arrestin binds does all trans retinal dissociate!!
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E* shutoff
RGS = regulator of G protein signaling
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Regenerate cGMP by guanylate cyclase
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All kinds of feedback to make recovery faster if high light levels : Ca2+ signalling
#1 shut off R* #2 shut off E*
#3 make cGMP
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Measure membrane current in rod photoreceptor
Current decreases with light= channels close
Electrophysiology
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Circulating current decreases
• As flash more light, channels close and current drops
• Then current recovers as channels open again
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How many ways do rods and cones differ?
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Rods and cones differ1. Morphology of outer segment
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Disks are distinct in rods
• Special proteins in rim help disks to formPeripherinRom-1ABCR/Rim
moves retinal across membrane
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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
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Rods and cones differ
3. Location and number
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Suction pipette measures membrane current
In response to light: current decreases because channels close
Rods and cones differ #4 Electrophysiology
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Rods and cones differ in how respond to light
• RodsHigh sensitivitySaturateSlow
• ConesLow sensitivityBig dynamic rangeFast
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Rods and cones differ #4. Electrophysiology
Circulating current decreases
NOTE – decrease in current is up on y axis
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Relative sensitivity
10-4 10-3 10-2 0.1 1 10 102 103 104 105 106
Photons / sec
rods
conesRods can detect single photons
Rod saturationAbsolute threshold
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Retinal isomerization
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Rods and cones differ5. Phototransduction pathway
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Phototransduction proteins
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Phototransduction proteins
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LWS
RH2
SWS2
SWS1
RH1
What does this tree tell us about rod and cone opsins?
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Conclusion #2• Rod opsins evolved from cone
opsins
LWS
SWS1
SWS2
RH2
RH1
Rhodopsin is Greek for rose + vision refers to color of pigment when look at dissected retina
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Evolution of rods from cones
Cones only
Rods and cones
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By looking at chromosomes containing opsin genes can see they came from duplicated chromosomes
SWS1 = OPN1SWLWS = OPN1LWRH1 = RHO
Chromosomal duplication and then tandem duplication
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Rod and cone Gα protein on duplicated chromosomes
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PDE genes also on duplicated chromosomes
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Phylogenetic test #1: rod - cone splitMammals
Birds
Amphibians
Fish
Mammals
Birds
Amphibians
Fish
Rod
Cone
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What does all this suggest about rod and cone pathways??
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Comparison of rod and cone electrophysiology and pathway
• Rod 100-1000x more sensitive
• ConeLarger Ca2+ currentFaster NCKX?Less gain in Gα activationFaster Rh* deactivation
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Hypothesis #2
• Differences in electrophysiology are the result of differences in some of the phototransduction protein sequences
Certain proteins are key - which ones?
Take Genomics of sensory systems BSCI338c next spring
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Summary
• Photoreceptors work a bit differently from other neuronsRods tailored to low light levelsCones tailored to bright light levels
• Rod and cone pathways are result of whole duplicate whole genome duplicationOccurred > 450 MY (before fishes diverged)
• Proteins in each pathway can then be tailored for rod or cone function