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Pharmacology of Ion Channels > Techniques > Flashcards

Techniques Flashcards

1
Q

TIRF

A

Total Internal Reflection Microscopy - used to visualise single vesicles during approach + fusion with the PM; fluorescent objects ~300nm of the PM

Need:

  • Fluorescently labelled/loaded vesicles
  • Fluorescence microscope equipped for TIRF imaging

Controls = Ca-dependent fluorescence

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2
Q

Flash photolysis

A

Caged Ca - EGTA-photosensitive cage
Whole-cell patch clamp
EGTA = calcium cheater - photosensitive

UV flash = uniform [Ca] elevation, measure exocytic events

BUT - recordings do not last = poisons cells

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3
Q

Membrane capacitance studies

A

Indirect measurement of vesicle exocytosis

Capacitance determines how quickly a cell’s Vm responds to a change in current

Surface area directly proportional to capacitance = increase s.a., increase capacitance
SCV ~ 2.5 fF

Voltage-Clamp - can see capacitative transients = exocytic events

Remember to use flash photolysis —cannot be measured if there is changes of conductances occurring in the membrane (ie. activation of VGCCs to stimulate exocytic events)

Problems with whole-cell patch clamp diluting IC contents

Net capacitance is a sum of exo and endo events BUT endo slower time course!

Net capacitance sum of ALL fusion events - even empire vesicles!

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4
Q

Amperometry

A

Electrochemical detection of catecholamine release in real-time
Dopamine, adrenaline, noradrenaline - oxidised at positive potentials

Carbon fibre electrode charged to positive potential - close to release sites (~5um) - detection of catecholamine release in ‘diffusion spikes’

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5
Q

Microamperometry

A

Detection of single secretory vesicles in individual cells

Positively charged carbon fibre except 5um tip!
Close proximity required to pick up diffusion of transmitters when it is released via exocytosis
Tracer of release events = higher spike, larger area under diffusion curves, more catecholamine released

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6
Q

Concatenation

A

Tie peptides into a single, polypeptide chain = build concatenated tetrameric constructs
Place flexible, inert linking sequence between each subunit ie. glycine
Can include a C-terminal GFP-tag = helpful for the identification of positively transfected cells

Transfect HEK cells = biolistics, lipofection, virus ie. Sindbus

ie. Order of subunits in a compound
How many subunits are required to bind a drug (mutate binding site)

ie. How many subunits are required for a drug to bind
Can produce a channel with 1/2/3/4 mutations - therefore identify how many functional subunits are required for a drug to bind etc

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7
Q

Chromosome Walking

A

A form of positional cloning

Used to locate the position of a disease-causing gene along a chromosome

ie. Used to identify Kv1.4 in Shaker mutant (lacking channel)

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8
Q

Molecular docking

A

Key tool in structural, molecular biology + computer-assisted drug design; predict the predominant binding modes of a ligand with a protein of a known 3D-structure

Use crystal structures as templates
Allows structural modelling of the interactions between toxins and channels
Produce touch maps of interactions between channels + toxins

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9
Q

CFTR Experimental Conditions

A

Fibroblasts in Chinese hamster cells (no CFTR/cAMP-activated Cl- channel)
Transfect: biolistics, lipofection, viral (Sindbus virus)
Transfect: wild-type, F508del
Controls:
Neg - no transfection
Sham - transfection conditions w/ no DNA ie. gold bullet, plasmid with no DNA encoding channel

Whole-cell voltage clamp
Activate channels with Forskolin (activates cAMP pathways)

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10
Q

Cryo EM

A

Transmission electron microscopy at frozen temperatures - used to discover the Lasso motif in CFTR

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11
Q

How to experimentally see whether N-terminus/C-terminus is IC/EC?

A

Add epitope-tag to NTD
Express in a cell line
Add AB - if binds = extracelullar
Permeabilise + fix, add AB, if binds = intracellular

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12
Q

Experimentally see whether a drug is on basolateral/apical membrane or inside the cell

A

Biotin-label apical or basolateral proteins - see pattern of staining use a confocal microscopy (slices through the microscope); they are located either side of the tight junctions

AB against CFTR - compare pattern of staining to see where it is located!

Wild-type = CFTR staining matches apical membrane staining!

F508del = see a different staining pattern, most is inside the cell beneath the tight junction, but not the same as the basolateral staining – seeing CF inside the cell!

Supports the model that CFTR is misprocessed and trapped inside the ER - very little protein reaches the plasma membrane!

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13
Q

Cystic Fibrosis

Defective Stability

A

Iodide efflux technique

Blunt measure of the activity of channels; measures ionic flux
-Convenient way to assay the function of CFTR in a population of cells

GOOD

  • High through-put compared to patch-clamp
  • Less prone to artefacts compared to fluorescence-based assays

BAD

  • Do not know why the voltage is changing
    ie. If the single-channel conductance is increasing, if there are more channels in the membrane etc.

Could do super-ecliptic pH-luorin tagged CFTRs?
-Bleach membrane = see increase in fluorescence as receptors inserted into the membrane

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14
Q

Transfection + Controls

A 
Biolistics = gene-gun; gold bullet coated in DNA
Controls = untransfected, gold bullet with no DNA covered (make sure gold is inert)

Lipofection = DNA into a liposome, infect HEK cells

Viral transfection ie. Sindbus = place gene in a plasmid,

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15
Q

Genetic knock-out

A

Bad - compensation mechanisms - the phenotype might be comparable to wild-type due to up-regulation of other proteins

Maybe also try with pharmacological inhibition - no time for protein up/down-regulations BUT side effects due to lack of specificity!

Important to use a multi-disciplinary approach!!!
ie. Structural remodelling, mutagenesis, electrophysiology

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16
Q

Mutagenesis studies

A

Identify the importance of different residues in ligand binding

17
Q

Pharmacological modification

A

Always use more than 1 drug which targets the drug to validate effect is due to medication and not side-effects of the same drug!

18
Q

Ultracentrifuge sedimentation assays

A

Analyse conformational changes

ie. MyoVb binding Ca - folded-to-extended (absence/presence of Ca)

19
Q

Atomic Force Microscopy

A

Use to determine how many subunits are in a receptor
ie. P2X receptors, 5-HT3 receptors

Bind ABs, study architecture of the surface with AFM - by shining a laser! Plot against a binomial distribution curve!

20
Q

Cystic Fibrosis
Defective Regulation
Defective Conduction

A

Single-channel
Perforated inside-out recordings

Defective Regulation:
Measure: IBI, MBD, Po

Defective Conductance:
Measure single-channel conductance!

21
Q

FRAP/FLIP

A

FRAP = Fluorescence Recovery after Photobleaching

FLIP = Fluorescence Loss in Photobleaching
-See a more defined movement of receptors from one area to another

Requires pH-luorin tagged receptors

22
Q

GFP

A

Make sure function has not been altered

ie. Knock-out mice, add GFP-tagged protein, should restore function!

23
Q

James Hodge techniques - Sleep in Humans

A

Sleep labs can monitor sleep using polysomnography:

  • Electroencephalography (EEG) = tracks and records brain waves
  • Electrooculography = tracks and records eye movements; can track REM/non-REM sleep
  • Electromyography (EMG) = monitor and record electrical activity produced by skeletal muscles
24
Q

Sleep in flies

A

Automated actinograms = measuring the intensity of radiation in a fly with an IR beam - an optical sensor counts each time a fly crosses the beam

  • L/D = 12 hours light, 12 hours dark
  • DD

DAM = Drosophila Activity Monitor

  • 32 flies in an actinogram = recorded 20 times
  • Plots activity plots = output of body clock

GOOD:

  • V quantatitive
  • Simple + robust
  • High throughput assay good for screening drugs/mutations
25
Q

Sleep in mice

A

Wheel running activity of mice in L/D (12 hours/12 hours) and DD

26
Q

Sleep in flies = SHY

A 
SHY = synaptic homeostasis mechanisms
Awake = syanptogenesis, LTP
Asleep = synaptic pruning, LTD

Structural = number + size of spines
- GFP-tagged actin, super-resolution microscopy (STED), count spines
Molecular = number of post-synaptic AMPARs
- Dissociated neurones
- Immunolabelling
- Glutamate-uncaging experiments = measure glutamate sensitivity
Electrophysiology = EPSP size, frequency of AP firing
- Hippocampal slices, stimulate CA3 + measure in CA1

27
Q

Luciferase assays

A

Study gene expression at the transcriptional level

+ = quantitative (extreme sensitivity therefore quantitative of small changes in transcription), convenient, cheap

Luminescent luciferase = put down-stream of clock controlled genes in an expression vector
Transfect cell line
Real-time bioluminescence recording of gene expression

28
Q

Quantify protein expression

A

Western blots with scanning densiometry

29
Q

Quantify gene expression

A

qRT-PCR

30
Q

James Hodge - The Clock

A

MICE

Actigram = wheel-running activity/fly w/ IR beam - shows activity

Conductances of channels = BK, SK, Kv1.3 L-type Ca channels

Bioluminescence luciferase reporter assays = period (per2), BMAL2

31
Q

Drosophilla learning centres

A

Mushroom body

Antennal lobe

32
Q

Flies = testing memory

A

Olfactory shock associate memory - mushroom body
- Give an odour at the same time as an electric shock
- Give a different odour without an electric shock
- T-maze = show learning by avoiding the shock-associated odour
Count number of flies in each area of the maze (different areas contain the different odours)

Performance index = (correct - incorrect)/total number of individuals

*** Requires dopamine!!!

33
Q

Astrocytic markers

A

EAAT 1/2

34
Q

GABAergic markers

A

GAD67

35
Q

Neuronal marker

A

MAP2, MCT2, NeuN

Pharmacology of Ion Channels (9 decks)

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