11th Nov - Phototransduction

Question 1

Outline the signalling pathway of rhodopsin in the dark state

Answer 1

Rod outer segment [cGMP] is high –> activation of cGMP regulated cation channels –> High intracellular calcium levels (about 500nM) –> Partially deplolarised pm (about -40mV) –> Tonic glutamate release

Question 2

What type of ligand is 11-cis-retinal?

Answer 2

An inverse agonist, holding the receptor in an inactive conformation

Question 3

What change does light cause to 11-cis-retinal?

Answer 3

Ionisation creating 11-trans-retinal –> ligand agonist

Question 4

What is the signalling pathway of rhodopsin in the light state?

Answer 4

Rh* –> Galphat –> PDE6 activation –> decrease in cGMP and increase in 5’GMP –> cGMP cation channels closing –> membrane hyperpolarisation

Question 5

How does Galphat promote PDE6?

Answer 5

Galphat sequesters inhibitory gamma units

Question 6

Why must rhodopsin recovery be so efficient?

Answer 6

We have a tethered ligand therefore the system must be actively turned off as to re-energize rhodopsin a new 11-cis-retinal must replace 11-trans-retinal which is a slow process

Question 7

Outline the recovery process of rhodopsin

Answer 7

1. Inactivation of rhodopsin through multiple serine phosphorylations by GRK1, creating a high affinity binding site for arrestin 1. The all-trans-retinal is released from opsin and repleaced by covalent attachment of a new molecule of 11-cis-retinal. (In order to do this the 11-trans-retinal must leave the rod-outersegment and enter the retinal pigment epithelium where it is converted to 11-cis-retinal by RPE65 and LRAT.)
2. Inactivation of Galphat -GTP - intrinsic GTPase activity of transducin (galphat) accelerated by RGS9-1 (GAP)
3. Resynthesis of [cGMP] - on illumination [Ca2+] decreases –> Ca2+ dissociates from GC activating protein (GCAP) –> Increased rate of cGMP synthesis

Question 8

How was RGS-9 identified as the GAP for galphat?

Answer 8

Inactivated both alleles of RGS9-1 in mice –> RGS9 KO
RGS9KO had slow GTP hydrolysis
In RGS9 +/+ cells PDE gamma sped up hydrolysis
In RGS9KO cells PDE gamma had no effect

Question 9

How do rod cells adapt to altering light levels?

Answer 9

Changes in the basal calcium level in the rod outer segment
Changes in the basal phosphorylation status of rhodopsin
Translcoation of key phototransduction components between the rod outer and inner segment

Question 10

What are the calcium sensitive proteins associated with Calciu regulation of photo-adaptation?

Answer 10

Recoverin
GCAP
Calmodulin

Question 11

How does Recoverin help with phot-adaptation?

Answer 11

In it’s calcium bound form it binds tightly to GRK1 preventing it from phosphorylating Rh

Question 12

How does GCAP help with photo-adaptation?

Answer 12

Its inactive in its Calcium bound state. When calcium levels decrease it becomes free and activates GC

Question 13

How does calmodulin help with photo-adaptation?

Answer 13

In it’s calcium bound form it binds and inhibits the cGMP regulated cation channel

Question 14

How does rhodopsin phosphorylation alter with light levels?

Answer 14

GRK1 can phosphorylate rhodopsin at three different positions - S343, S338, S334. When it is fully phosphorylated GRK1 will bind.

With increasing light levels phosphorylation of rhodopsin increases

Question 15

Which components translocate into the rod outer segment on prolonged light exposure?

Answer 15

arrestin

RGS9-1

Question 16

Which components translocated out of the rod outer segment on prolonged light exposure?

Answer 16

Transducin

Recoverin

Question 17

Why do components of the rhodopsin signalling cascade translocate into/out of the rod outer segment in response to a prolonged light exposure

Answer 17

It alters the ability of the phototransduction cascade to amplify and shut off signalling

Question 18

What is the experimental evidence for recoverin translocation from the outer–> inner segment on light exposure?

Answer 18

Strussel 2005
Took 22 5µM sections of a rod cell and performed western blots of each section using 2 markers: rhodopsin (A marker for outer segments) and cytochrome oxidase (A marker for inner segments). Found that recoverin was at a higher concentration in the outer segment in the dark adn in the synaptic terminal in the light
Rhodopsin and Cytochrome oxidase distribution did not alter

Question 19

Why does transducin concentration decrease in the rod outer segment in high light intensities?

Answer 19

As the rod cells are sensitive to light, this allows vision from rod cells at bright light intensities

Question 20

How do the rhodopsin signalling components translocate in varying light intensities?

Answer 20

No one knows

Active transport - would be rapid and unidirectional but could easily be overloaded with the number of translocating proteins

Diffusion - is limitless and takes about 3.5 minutes (rapid enough to explain the light driven translocation of transducin and arrestin)