Phototransduction Cascade quick review Single Cell responses

Electrophysiology of Photoreceptors (from counting photons in starlight to the blazing sun snowy slopes)Electrophysiology of Photoreceptors (from counting photons in starlight to the blazing sun snowy slopes)

Phototransduction Cascade quick review

Single Cell responsesCurrents, voltages transmitter release

Rod and cone response differences

RPE Cells

Photoreceptors

Müller Cells

RPE Cells

Photoreceptors

Müller Cells

Brett Gerwin MSII

Salamander Rod and Cone cellSalamander Rod and Cone cell

Synaptic region

Ellipsoid

OuterSegments

Light is the ligand that triggers activation of the enzyme.

Ca2+

Rods AND ConesRods AND Cones

Circulating current between the OS and IS in the dark partially depolarizes the cells.

Light triggers HYPERPOLARIZATION and decreased transmitter release. Glutamate is the neurotransmitter.

Biochemical cascade initiated by absorption of one photon by chromophore (11-cis retinal).

Activated opsin acts as an enzyme. Rhodopsin and cone opsins are the classical G-protein couple receptor (GPCR).

Opsin activates transducin, which activates phosphodiesterase (PDE). Activated PDE destroys cGMP

cGMP is the 2nd messenger that keeps cation channels open

Detecting A Single QuantumDetecting A Single Quantum

In rods a single photon generates an electrical signal that is sufficiently above the noise so as to be reliably detectable.

Photocurrents are graded responses to lightthat changes membrane voltage which in turn drives neurotransmitter release

Photocurrents are graded responses to lightthat changes membrane voltage which in turn drives neurotransmitter release

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Rod sensitivity is high at a cost of speed, slow temporal sensitivityRod sensitivity is high at a cost of speed, slow temporal sensitivity

Toad rod recording at 20°C

Two cell types; two functionsTwo cell types; two functions

Rod & Cone OS differ physically Rod & Cone OS differ physically

Rods vs. ConesRods vs. Cones Rod shaped OS Separate discs

Slower pigment regeneration (renewal)

Synaptic ending is small round spherule with few ribbons

Connect only to On-type, rod bipolars

Cone shaped OS Fused discs, continuous

with extracellular space Pedicle shaped synaptic

terminal with More ribbon synapses (20)

Connects to many types of BOTH on & off Bipolar cells

Rods vs. Cones: Kinetic & Sensitivity DifferencesRods vs. Cones: Kinetic & Sensitivity Differences

Where do these differences arise?Where do these differences arise?

Rods vs. Cones BiochemicalRods vs. Cones Biochemical Very stable visual

pigment Greater biochem gain Slower responses Lower Ca++

permeability through cGMP channel

Saturation Limited operating range

Less stable visual pigment Lower sensitivity(gain) Faster temporal response Greater Ca++ permeability

through cGMP channel CONES NEVER

SATURATE to steady light.

10x faster PDE inactivation.

Chipmunk Rod:

I1/2 = 200 photonµm -2

Sf = 0.090 pA-photon -1µm2

ttpeak = 100 msec

Ti = 90 msec

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A look at rod responses: the #’sA look at rod responses: the #’s

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Chipmunk Cones:I1/2 = 7,000 photon- µm-2

Sf =0.0005 pA-photon-1µm2

ttpeak = 50 msec

Ti = 40 msec

Cone responses: the number’s

Chipmunk Cones:

I1/2 = 7,000 photon- µm-2

Sf =0.0005 pA-photon-1µm2

ttpeak = 50 msec

Ti = 40 ms

ROD/CONE Ratios

Chipmunk Rod:

I1/2 = 200 photonµm -2

Sf = 0.090 pA-photon -1µm2

ttpeak = 100 ms

Ti = 90 ms

Rod/Cones:

I1/2 : 1/35

Sf : 1/180

ttpeak : 2

Ti : 2.25

Inactivation steps control sensitivity and timingInactivation steps control sensitivity and timing

ROD and CONE transduction are different! 2x timing 50 to 100x in sensitivity

Although the specific details of the differences is not yet known. . . . Kinases for phosphorylation of R* differ The cGMP gated channels are different GCAP proteins that are Ca++ sensitive feedback signal are

different Inactivation of PDE* by RGS9 are probably different (Cones have

10x rod levels of RGS9)

Phototranscduction CascadePhototranscduction Cascade

Photon of light generates R* Stage 1: R* collides with G protein (both on the membrane

disk) (500 to 800 fold amplification) G-GDP : R* promotes exchange of GTP for GDP G protein splits to become active G-GTP and the G

Stage 2: G-GTP collides with and attaches to the enzyme PDE () complex dislodging an inhibitory unit (PDE) Gain factor 1. The G-GTP- PDE complex greatly enhances PDE activity

Stage 3: activated PDE hydrolyzes cGMP -> 5’ GMP Gain factor 6-50

TOTAL GAIN about 5000 cGMP destroyed, 1,000,000 Na/Ca ions excluded from outer segment of rod photoreceptor outer segment.

Phototranscduction CascadePhototranscduction Cascade

RGS9/G5/R9AP

Cyclic activity of enzymes in CascadeCyclic activity of enzymes in Cascade

Increasing RGS9/G5/R9AP proteins 25 fold alters the rod response.Increasing RGS9/G5/R9AP proteins 25 fold alters the rod response.

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Control eGFP

Calcium FeedbackCalcium Feedback

Light closes the outer segment cation channel reducing influx of Ca2+, a potent feedback signal in phototransduction.

Calcium FeedbackCalcium Feedback Guanylate cyclase replaces the cGMP

to reopen the channel to repolarize the membrane back to resting levels. Cyclase activity is cubically dependent on

Ca2+

Calmodulin is a calcium binding protein that interacts with the cGMP channel to modulate cGMP binding affinity.

Recoverin is modulated by Ca2+ and is part of the rhodopsin recovery pathway.

Shutting off PhototransductionShutting off Phototransduction

The size of the signal (the gain) depends on how long the cascade remains active.

Each step of the cascade must be reversed to shut off the signal (Enzymes inactivated) SPEED vs. SENSITIVITY

The inactivation of PDE* depends on a complex of 3 proteins: RGS9, G5 & R9AP Rod vs. Cone gain may depend on PDE* inactivation

rate and RGS9 amounts.

Inactivation steps control sensitivity and timingInactivation steps control sensitivity and timing

ROD and CONE transduction are different! Although the specific details of the

differences is not yet known. . . . Kinases for phosphorylation of R* differ (GRK1 &

GRK7)

Inactivation of PDE* by RGS9 are probably different (Cones have 10x rod levels of RGS9)

Cone channel admits more Ca2+, providing a faster feedback signal to

Guanylate cyclase (replenishes cGMP to open channels, GCAP)

Recoverin (inhibits Kinase that shuts off R*) Through Calmodulin acting on channel itself (increase

K1/2)

Electrical responses can shape visual behaviorElectrical responses can shape visual behavior Simple - if the photoreceptors can’t see it,

How can the visual system? At threshold the rods are counting photons at

the rate of 1/85 minutes!!! Summation at the bipolar cells

What is adaptation?What is adaptation?

When a constant stimulus results in a variable response. Usually smaller

AdaptationMeasures of toad rod sensitivity on various backgrounds

Measures of toad rod sensitivity on various backgrounds

Toad Rod recording

Later Yau and colleagues find some adaptation in monkey rods

Later Yau and colleagues find some adaptation in monkey rods

Human rod adaptation:Follows Weber’s law

Human rod adaptation:Follows Weber’s law

Minor changes in kinetics accompany light adaptation.

Adaptation in rods may take several seconds to manifest

Adaptation in rods may take several seconds to manifest

Rod photoreceptors do adapt

-very slowly

-to only a limited degree

Cone Adaptation begins within 1 sec

Cone Adaptation begins within 1 sec

Primate cones follow Weber’s law with background light

Cone responses on backgroundCone responses on backgroundThe presence of a background light shifts the operating range of the cone: so increments and decrement can be encoded.

Cones adapt to center their operational range near 50% VmaxCones adapt to center their operational range near 50% Vmax

Background light causes the cones to shift their operating range

Background light causes the cones to shift their operating range

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Background light induces undershootBackground light induces undershoot

Dark Dark

Ib = 6.08 log photons/µm2S

Temporal shape of the response can influence behavior as well.

Human flicker sensitivity shows a transition from low-pass to band-pass filtering with background lights.