Lecture 14: Optogenetics Flashcards
What are opsins?
Membrane receptors that respond to light.
What class of receptor are they? What is their structure?
GPCRs. 7TM domains
Which part detects light?
11 cis retinal
What 2 superfamilies are opsin genes divided into?
Type 1 are microbial type opsins. Type 2 are animal type opsins
What are rhodopsins?
Expressed in vertebrate rod photoreceptors
What is the difference between Type 1 and Type 2?
Low sequence homology, structural divergence, different chromophore chemistry, different signal transduction mechanisms.
What do type 1 include and how do algae use it?
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
What do the channels transport?
BR does protons, HR does Cl- and ChR does Na+, K+, Ca2+ and protons
What are type 2 responsible for?
In eukaryotes for vision and cardiac rhythm.
How many different type 2 are found?
Over 1000
What is rhodopsin?
A light sensitive GPCR with a 40 kDa apoprotein, opsin and a chromophore 11 cis-retinal
Where is rhodopsin found?
The outer segment of rod cells in the retinal of the vertebrate eye
What happens when a photon hits rhodopsin?
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.
What is the photo cycle comprised of?
a series of proton transfer reactions until the proton moves from the intracellular to the extracellular space.
Describe how BR is activated.
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)
What are conserved in type 1?
The AA involved in proton translocation
What is the trigger for all structural rearrangements?
Photoisomerised retinal
How are opsins used in neuroscience?
Can target individual neurones and integrate opsins into them to regulate the activity and study the impact on the whole of the brain.
What causes the action potential, how is this achieved?
ChR transfer Na+ and help cause action potential. Is put into the neurone, light shone, conf change, channel opens and Na+ moves into membrane.
How can you recalibrate the neurone?
Can use HR for Cl- to recalibrate the neurone.
How can this be applied to hearts and glucose levels?
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.
What are some of the challenges?
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.
What has optogentics been used to study?
sensory pain perception, decision making, social interactions, feeding behaviours, regulating motor function in parkinsons and targetting neurones for post traumatic stress disorder.
