Module 13 - optogenetics, dues and radioligands Flashcards
fluorescence how
Absorb high energy light
Then emit light at LONGER wavelength at lower energy
why reporter dyes better than electrophys
- not invasive, no cells destroyed
- electrophys has limits to what we can patch onto
- reporter dyes can do multiple cells at a time
what are 3 types of calcium sensitive dyes
low affinity = excited by UV light
intermediate affinity = excited by UV light
high affinity = excited by visible light
how does fura-2 bind calcium
has 4 COOH- groups
specific to divalent ions ie Ca2+
how does fura-2 change after Ca2+ bound
causes distortion
cahnges excitation wavelength
how would you use fura dye to measure conc of Ca2+
excite sample at 340nm then 380nm
measure emmision at 510nm for each
- 340 gives conc of Fura2 that has Ca2+ bound to it
- 380 gives Fura2 that isnt bound to Ca2+
Then take the ratio: emm from 340/ emm from 380
what 2 things are affected when Ca2+ added to a sample
the emmision peak increases in magnitude, but remains at 510
the absorption shifts from 380 to 340
what changes Fura from polar to non-polar
esterify it with Acetoxymethyl Ester (AM ester)
to make Fura-2 AM
problem with Fura 2 AM
crosses the memb but can bind Ca2+ cuz all COOHs are esterified
so in the cell, esterases act to remove AM
2 main advantages of these dyes
look at ion fluxes in real time
show spatial distribtuion of ion flux
disadvantage of dye
lacks specificity for cell type
What are GINAs
Genetically encoded indicators of neuronal activity
E.g. GCamp
Advantage of GINAs
can be targeted to individual cells and tissues
Can have temporal control
Structure of GCamp
a fusion protein
Green fluorescent protein bound to calmodulin and M13
what is FRET
flurescence resonance enrgy transfer
what position do molecules need to be for FRET to occur
when 2 molecules in close proximity
e.g. when ligand binds to receptor and the wavelength emitted by one will trigger the next to emit its wavelength
name of non slective light gated cation channel used in optogenetics
channelrhodopsin 2 (ChR2)
responds to blue light
causes influx of cations= depolarisation
name of chloride pump powered by light
halorhodopsin (NpHR)
responds to yellow light
influx of Cl- = hyperpol
what is ChR2 H134R
mutated ChR2 channel
his at 134 position to arg
makes channel more conductive
what molecule do both channels have built in that detects the light
retinal cofactor
3 ways to get the channel/pump protein into neurons
plasmid ie transfection
transgenic animals
viral vectors
what is used to deliver the light in a cell culture vs a whole animal
cell culture = jsut normal LED light with collimator
animal = fibre optic probe
what is the human, light sensitive GPCR
rhodopsin
interacts w/ Transducin (Gt) = decreases cGMP
name of technique when optogenetics used to control GPCR pathways
OptoXRs
how to make chimeric GPCRs that opto can act on in the brain
transmemb and extracellular domains of rhodopsin + intracellular domain of any desired GPCR
