GABA - BS42025 Flashcards
Most commonly used IV induction agent?
Propofol
Pioneer of Anaesthesia
Sir James Young Simpson 1811-1870.
Identified chloroform as clinical anasthetic
General anaesthetics are stucturally diverse
Hard to know that they’d bind to the same structures
Examples: etomidate, midazolam, propofol, halothane, alphaxalone
Lipid soluble - will dissolve in neuronal membrane
Experiment: do general anaesthetics act non-specifically via the membrane?
By Meyer and Overton
Used tadpoles, measured how much aneasthetic it took for 50% of animals to lose the ability to right themselves again (EC50)
For most compounds could predict it, by calculating the lipid solubility of drugs (by how well it dissolved in olive oil)
Increased potency = increased solubility
Positive correlation
Anaesthetics thought to act by perturbing membrane strcuture
No longer a positive correlation after C9 chain length in alcohol - still increased solubulity, no longer increased potency
Anaesthetics exist with potency not predicted by lipid solubility
Anaesthetic enantiomers - same solubility, same ability to perturb membrane, different potency
Researchers in London showed that anaesthetics can bind to a pure protein preparation and change what it did (fire fly luciferase) - so which proteins did it bind to?
Transmitter-gated ion channel family of receptors
Cys-loop receptor (nicotinic ACh) superfamily - pentamer
Glutamate receptor family - tetramer
Cys-loop receptor family
Nicotinic ACh receptor family
5-HT3 receptors
GABAA receptors
Glycine receptors
- All pentamers (5 subunits)
- 4 membrane spanning subunits (TM2 is part of ion channel)
Glutamate receptor family
NMDA Receptors
AMPA Receptors
Kainate Receptors
- Tetramers
- Excitatory neurotransmitter
- 3 transmembrane spanning subunits and 1 re-entrant loop (part of ion channel)
Using frog eggs to express human brain receptors
Xenopus laevis - use oocytes (have large nucleus)
Inject human DNA/RNA into nucleus - makes the proteins and traffics them to cell surface
Record activity of receptors using 2 point voltage clamp
Voltage electrode will detect something like -60 mv
Current electrode will pump in current to prevent cell from hyperpolarising or depolarising
Adding GABA activates receptors - Cl- will go out (usually in neurons goes in, but these oocytes have high extracellular CL1-concentrations)
Measure current needed to keep voltage at -60mV
Use GABA concentration that produces about 10% of max response
Add propafol
Reapply same conc of GABA - hugely increased response
Effect of etomidate on different receptors when tested in Xenopus laevis oocytes?
GABAA - increased effect of GABA
Glycine - none
AMPA/Kainate - none
NMDA + Glycine - none
Alpha4Beta2 nicotinic - none
5HT3 - none
Effect of propofol on different receptors when tested in Xenopus laevis oocytes?
GABAA - increased effect of GABA
Glycine - enhanced glycine slightly
AMPA/Kainate - none
NMDA + Glycine - none
Alpha4Beta2 nicotinic - caused some inhibition
5HT3 - very slight inhibition
Effect of alphaxalone on different receptors when tested in Xenopus laevis oocytes?
Steroidal anaesthetic
GABAA - increased effect of GABA
Glycine - none
AMPA/Kainate - none
NMDA + Glycine - none
Alpha4Beta2 nicotinic - inhibit nicotinic receptor function
5HT3 - none
Betaxalone, similar structure o alphaxalone, had same effect on nicotinic receptor but not GABA receptor - is not an anaesthetic
Effect of pentobarbitone on different receptors when tested in Xenopus laevis oocytes?
GABAA - increased effect of GABA
Glycine - none
AMPA/Kainate - some inhibition
NMDA + Glycine - slight inhibition
Alpha4Beta2 nicotinic - inhibits nicotinic receptors a lot
5HT3 - slight inhibition
Are the receptors mentioned in regard to the Xenapus laevis oocytes inhibitory or excitatory?
GABAA - inhibitory
Glycine - inhibitory
AMPA/Kainate - excitatory
NMDA + Glycine - excitatory
Alpha4Beta2 nicotinic - excitatory
5HT3 - excitatory
Effect of ketamine?
Little effect on GABA A receptors, inhibits NMDA receptors
Enantioselective action of etomidate
R + etomidate (also known as hypnodate) - much more potent
S - etomidate
S- needs about 10x higher concentration to get tadpoles or mice to lose the ability to right themselves
Have equal ability to dissolve into and perturb membrane
Ability to modulate and activate GABA is much higher in the R than S form
GABAA Receptor summary
- Major inhibitory receptor in mammalian brain
- GABAAR inhibitors (eg bicuculline (competitive antagonist), picrotoxin (blocks associated ion channel)) are proconvulsant and anxiogenic
- Drugs that enhance GABAAR function (eg diazepam) are anticonvulsant, anxiolytic, sedative, and analgesic
Roughly how many GABAA Receptor isoforms does the mammalian CNS express?
20-30
Heterogeneous distribution
About 19 different subunits that make up the isoforms
Largest amount: alpha1 beta gamma2
General anaesthetics produce a number of behaviours including…
sedation, immobility in response to surgical incision, unconsciousness
Oocyte recordings to show different subunits react to different anaesthetics
Oocytes injected with cDNAs encoding for alpha6, beta2, and gamma2 subunits
1 micromole of GABA activates the receptors
Same things as initial discovery: Cl- leaves, after adding 10micromoles of etomidate response greatly increases
Changing beta2 to beta1, only a small increase is seen
(Etomidate also likes beta3)
Beta subunit selectivity of etomidate
Beta1 not susceptible to etomidate
Beta2 is susceptible to etomidate
Beta2/1 chimeras (extracellular loop beta2 (n-terminal), rest beta1) - activated by gaba, but not really affected by etomidate
Beta1/2 chimeras (extracellular loop beta1 (n-terminal), resta beta2) - large effect to etomidate. critical region in TM2 - at position 265 should be an asparigine (in beta 2 and 3), but is serine in beta1.
Is serine is changed to asparagine in beta1, etomidate will largely increase gaba response
Done by Weir, Mitchell, Lambert in 2017
Membrane play a role in anaesthetic?
Anaesthetic may concentrate in membrane close to transmembrane binding site located between beta-alpha subunit interface
Olsen, 2018
Binding of etomidate/propofol
Etomidate binds at the β-α interface with the phenyl ring packed against N265 of the β2 subunit.
Propofol binding overlaps with etomidate, but is smaller so makes fewer contacts with the receptor
When GABA is bound all 5 interfacial sites are open
Etomidate binds - gamma2beta2 and alpha1beta2 sites close
Propofol binds - all sites remain open
Kim, 2020
How were mice engineered for etomidate testing?
Beta2 N265S (asparigine to serine) - reduced etomidate sensitivity
Beta3 N265M (asparigine to methianine) - reduced etomidate and propofol sensitivity
LORR by etomidate is reduced in both the beta2 N265S and beta3 N265M. Still acts in that instance
Beta2 N265S Mouse
Locomoter activity in activity cage - counts when mouse crosses laser beams
WT: at 1mg/kg of etomidate, mouse starts to move less, at 12.5 mg/kg mouse becomes unconsciouss. Lots of slow wave sleep pattern on EEG
Mutant: etomidate doesn’t really affect up until 7.5mg/kg, higher than 10 mouse becomes unconsciouss. SLow wave sleep pattern greatly reduced
Sedative effects at lower doses blocked, can still cause hypnosis or unconsciousness at higher doses
Loss of righting reflex (LORR)?
A surrogate measure of unconsciousness “anaesthesia” in humans
When put on its back, it can no longer right itself
Quite a correlation between the anaesthetic dose required to cause a loss of consciousness in 50% of humans (EC50) (failure to respond to verbal commands and the dose required to produce LORR in rats and mice
Tail withdrawal reflex in mice mutated for etomidate?
Immobilising effects of etomidate and propofol are blunted in the beta3N265M mouse, but not the beta2N265S mouse
WT - withdrawals tail
WT + etomidate - does not withdrawal
Beta3N265M + etomidate - withdrawals tail
Beta2N265S + etomidate - does not withdrawal tail
Beta3 densely expressed in spinal cord - possible locus of reflex
Beta2N265S summary
With etomidate:
Hypnosis in part reduced (can still go unconsciouss)
Sedation completely impaired
No longer causes hypothermia (usually anaesthetics do)
Beta3N265M summary
With etomidate and propofol:
Hypnosis in part reduced
Immobility is reduced
Respiratory depression no longer occurs
Etomidate Summary
PAM of GABAARs
Highly selective for beta2 and beta3, not beta1
Beta2 and beta3 governed by asparigine residue at 265, and beta1 by serine residue at 265
What technique is used to record GABA-ergic activity from single neurons in an in vitro brain slice?
Patch clamp technique, recording in the whole-cell voltage-clamp mode
How does whole cell patch-clamp voltage recording work?
Recording electrode positioned over nueronal cell body, eletrode gently touches cell surface, small amount of suction formes omega shaped piece of membrane drawn up into pipette, more severe suction breaks membrane, patch electrode contains physiological salt solution which now enters cell, can pass current through electrode to maintain membrane potential at roughly -60/-70mV (for neurons).
Some of the contents ie kinases of the neuron may be pulled up into the pipette.
Patch clamp attached to patch clamp amplifyer to record voltage changes
What does ‘in vivo’ mean?
Within a living organism
What does ‘in vitro’ mean?
In an atrificial environment
Recording phasic and tonic GABAAR-mediated inhibition in brain slices
Recorded from cell body
Whole cell patch-clamp, voltage clamped at -60mV
Saline solution containing kynurenic acid (2 mM - blocks ionotropic glutamate receptors), tetrodotoxin (0.5 microM - to block presynaptic action potentials)
Looking at single vesicle of GABA (miniature inhibitory postsynaptic current (mIPSC)
See inward currents - appear as upside down triangles (this is upon release of a vesicle of GABA, which activates postsynaptic receptors and transports Cl- in)
When the triangle goes back down, this is when GABA channels are closing. PAMs prolong how long they stay open
-> This is phasic inhibition (IN synapse
Tonic inhibition
Less prone to desensitisation
Activated by low levels of GABA that has spilled over from synapse
Creates slow tonic current - constant open and closing of receptors creates ‘noise’
Know it’s from GABA because applying bicuculline (GABA antagonist) makes small tonic current appear
GABA and Thalamus
Thalamus known as gateway to the cortex
Ventrobasal nucleus (VB) receives inhibitory GABA input from nucleus reticularis (nRT - contains sheets of GABAergic neurons)
VB sends excitatory (glutamate) connections to the nRT and cortex
Cortex sends excitatory (glutamate) connections back to the nRT and to the VB neurons
Thalamocortical VB Neurons and patch-camp recordings
Synaptic GABAA receptors (alpha1beta2gamma2)
Extra synaptic GABAA receptors (apha4beta2delta) - benzodiazepine insensitive bc alpha4 and bc delta
Beta2 are etomidate sensitive
Effect of etomidate on synaptic and extrasynaptic inhibition of thalamocortical neurons
WT - etomidate sedates
Beta2N265S - etomidate does not sedate
WT - etomidates elongates synaptic current, and tonic current by about 86%
Beta2N265S - etomidate had no effect on synaptic or tonic current
