Lecture 14: Optogenetics

Question 1

What are opsins?

Answer 1

Membrane receptors that respond to light.

Question 2

What class of receptor are they? What is their structure?

Answer 2

GPCRs. 7TM domains

Question 3

Which part detects light?

Answer 3

11 cis retinal

Question 4

What 2 superfamilies are opsin genes divided into?

Answer 4

Type 1 are microbial type opsins. Type 2 are animal type opsins

Question 5

What are rhodopsins?

Answer 5

Expressed in vertebrate rod photoreceptors

Question 6

What is the difference between Type 1 and Type 2?

Answer 6

Low sequence homology, structural divergence, different chromophore chemistry, different signal transduction mechanisms.

Question 7

What do type 1 include and how do algae use it?

Answer 7

Bacteriohodopsin (BR), halorhodopsin (HR), channelrhodopsin (ChR) and sensory rhodopsin (SR). Algae transduce light ti change flagella beating to direct them towards more light for more hpotosynthesis

Question 8

What do the channels transport?

Answer 8

BR does protons, HR does Cl- and ChR does Na+, K+, Ca2+ and protons

Question 9

What are type 2 responsible for?

Answer 9

In eukaryotes for vision and cardiac rhythm.

Question 10

How many different type 2 are found?

Answer 10

Over 1000

Question 11

What is rhodopsin?

Answer 11

A light sensitive GPCR with a 40 kDa apoprotein, opsin and a chromophore 11 cis-retinal

Question 12

Where is rhodopsin found?

Answer 12

The outer segment of rod cells in the retinal of the vertebrate eye

Question 13

What happens when a photon hits rhodopsin?

Answer 13

triggers conversion of 11-cis retinal to all trans retinal causing conf change and interacts with G protein, transducin. This activates cGMP phosphodiesterase which hydrolysis cGMP.

Question 14

What is the photo cycle comprised of?

Answer 14

a series of proton transfer reactions until the proton moves from the intracellular to the extracellular space.

Question 15

Describe how BR is activated.

Answer 15

After retinal has diffused into the binding pocket of the 7TM helix oval, it is covalently attached to a conserved lysine 296 residue of helix 7 by formation of a protonated retinal schiff base (RSBH+). Photon absorption initiates conf switch leading to discontinuous proton transfers involving Asp85, Asp96, Asp212 and Arg82 and a proton release complex (PRC)

Question 16

What are conserved in type 1?

Answer 16

The AA involved in proton translocation

Question 17

What is the trigger for all structural rearrangements?

Answer 17

Photoisomerised retinal

Question 18

How are opsins used in neuroscience?

Answer 18

Can target individual neurones and integrate opsins into them to regulate the activity and study the impact on the whole of the brain.

Question 19

What causes the action potential, how is this achieved?

Answer 19

ChR transfer Na+ and help cause action potential. Is put into the neurone, light shone, conf change, channel opens and Na+ moves into membrane.

Question 20

How can you recalibrate the neurone?

Answer 20

Can use HR for Cl- to recalibrate the neurone.

Question 21

How can this be applied to hearts and glucose levels?

Answer 21

Can control the contractibility of cardiac cells so can pace the heart of mice using light. Can regulate intracellular glucose levels to control release of insulin of B cells expressing ChR2.

Question 22

What are some of the challenges?

Answer 22

The mutant and endogenous gene will co exist so there won’t be complete control. Attachment of photoswitch requires that a cysteine site is freely accessible. Photoswitches require blue (380 nm ) light to transition the trans to cis state. Uv poorly penetrates the brain tissue and can result in cellular toxicity at high intensities.

Question 23

What has optogentics been used to study?

Answer 23

sensory pain perception, decision making, social interactions, feeding behaviours, regulating motor function in parkinsons and targetting neurones for post traumatic stress disorder.