Lecture # 14
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Transcript of Lecture # 14
Lecture # 14
Vertebrate phototransduction3/14/13
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
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
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
Phototransduction
• Transduction “the conversion of a signal from one form to another”
Photo - signal comes from light
Transduce – neural signal goes out
Typical neuron
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
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
-----
-----
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
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
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
Measure membrane current in rod photoreceptor
Current decreases with light= channels close
Electrophysiology
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
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
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
Rod structure – outer cell membrane with stack of discs inside
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
The players
• Visual pigment
Opsin protein surrounds 11-cis retinal
Combination absorb light
The players
• G proteinThree subunitsα binds GDP / GTPβγ binds inactive α
• Activates effectorFor vision it is α
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
cGMP gated ion channel
• Cooperatively binds 4 cGMP
• When cGMP is bound, channel is open
cGcG
cGcG
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
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
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.
G protein pathway in rods
Note the exchangerIt pumps Ca and K out and Na inAlways working
Phototransduction video
G protein pathway in rods
All the players work together to close channel and cause hyperpolarization
Phototransduction
• Relative proportion of proteins
Rhodopsin - 1000
Transducin - 100
PDE - 4
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
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
Circulating current• What happens if all
channels close??• Current goes to zero
as light level increases
How are they the same?
• Same – Gproteins sometimes; same Ca/ Na/K ions Graded response to change - Depolarization = neurotransmitter outputHyperpolarization = neurotranmitter decrease
How are rods weird / different from other sensory neurons?
• Signal = hyperpolarization• Signal = channels closing• Signal = less neurotranmitter
Turning off excitation-recovery
#1 shut off R* #2 shut off E*
#3 make cGMP
#4 reopen channels
R* shutoff
Note: Only after arrestin binds does all trans retinal dissociate!!
E* shutoff
RGS = regulator of G protein signaling
Regenerate cGMP by guanylate cyclase
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
Measure membrane current in rod photoreceptor
Current decreases with light= channels close
Electrophysiology
Circulating current decreases
• As flash more light, channels close and current drops
• Then current recovers as channels open again
How many ways do rods and cones differ?
Rods and cones differ1. Morphology of outer segment
Disks are distinct in rods
• Special proteins in rim help disks to formPeripherinRom-1ABCR/Rim
moves retinal across membrane
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
Rods and cones differ
3. Location and number
Suction pipette measures membrane current
In response to light: current decreases because channels close
Rods and cones differ #4 Electrophysiology
Rods and cones differ in how respond to light
• RodsHigh sensitivitySaturateSlow
• ConesLow sensitivityBig dynamic rangeFast
Rods and cones differ #4. Electrophysiology
Circulating current decreases
NOTE – decrease in current is up on y axis
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
Retinal isomerization
Rods and cones differ5. Phototransduction pathway
Phototransduction proteins
Phototransduction proteins
LWS
RH2
SWS2
SWS1
RH1
What does this tree tell us about rod and cone opsins?
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
Evolution of rods from cones
Cones only
Rods and cones
By looking at chromosomes containing opsin genes can see they came from duplicated chromosomes
SWS1 = OPN1SWLWS = OPN1LWRH1 = RHO
Chromosomal duplication and then tandem duplication
Rod and cone Gα protein on duplicated chromosomes
PDE genes also on duplicated chromosomes
Phylogenetic test #1: rod - cone splitMammals
Birds
Amphibians
Fish
Mammals
Birds
Amphibians
Fish
Rod
Cone
What does all this suggest about rod and cone pathways??
Comparison of rod and cone electrophysiology and pathway
• Rod 100-1000x more sensitive
• ConeLarger Ca2+ currentFaster NCKX?Less gain in Gα activationFaster Rh* deactivation
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
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