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

-----

-----

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