Iom channels Flashcards
Resting, reversal for sodium, reversal for potassium - potentials
-70mV
+60mV
-90mV
GABAa receptors are selective for…
Cl-, also permeable to some extent to HCO3-
Explain how the GABA receptor is not always an inhibitory receptor
when young, mammalian neuriones have a high Cl- conc inside (due to less KCC2 chellens to pump out Cl-). This means that the electrochemica potential is slightly more positive and reversal potential when just born is around –40mV, this means that in this situation, when GABAa channels are activated they make cells fire
As get older more KCC2 channels are there so Cl- levels inside neuron decrease to normal
When GABAa channels open they…
want to bring the cell back to the resting membrane potential. -> away from Action potential threshold (in adult mammals)
Explain how GABA receptor diversity is linked to interneuron diversity
take a slice of mammalian cortex and label cells in position and identity – you have a repeatable organization, and there are different subtypes of cells that synthesize and release GABA (inhibitory cells)
Features of GABA receptors
Ionotropic receptors of neurotransmitter GABA
Chloride permeable
Physiology
- Synaptic transmission (fast inhibition)
- Tonic inhibition
Drug types:
- Benzodiazepines (BDZs)
- Barbiturates
- Anaesthetics
- Neurosteroids
BZDs are one of the most prescribed classes of drugs for the treatment of insomnia, anxiety, and convulsions.
Diseases linked to GABA receptors
Epilepsy
Anxiety
Drug and ethanol abuse
Schizophrenia
Alzheimer’s disease
Pain
individual subunit of GABA
4 transmembrane domains
Large N-terminal extracellular domain
Small C-terminal extracellular domain
Multiple subunit structure of GABA
Pentamer
2nd transmembrane domain that comes together in the pentamer and forms the ion channel
Pentameric ligand-gated ion channel family (also nAChR, 5HT3R, GlyR)
subunit isoforms of gamma
a,b,y,delta,epsilon,pi,pho(p), theta (most common a1-6)
Most common type of GABA receptor (in terms of subunit isoforms)
Alpha1 beta2 gamma2
why are A5 GABA receptors interesting?
extrasynaptic, but modulated in dendrites – specifically in dendrires they seem to be localised
What is Gephryin
similar to PSD-95), an anchoring molecule that helps postsynaptic GABA receptors stay put
What is Radixin
scaffolds glutamate receptors so that they DONT go to synapses
Describe the different cells within the layers of the cerebral cortex and their GABA subtype
Layer 4 Basket cells (target α1 containing receptors) - well positioned to prevent action potential
Layer 2/3 Neurogliaform cells (target α5 containing receptors) - present almost everywhere in the dendritic tree
Layer 5 Chandelier cells (target α2 containing receptors) - at the axon – even better positioned than basket cells to prevent action potential firing
Extrasynaptic (α5βδ, α5βγ receptors)
Explain the binding sites of GABA receptors and how this was discovered
Bergmannet 2013
receptor is isolated, cloned and can be expressed specifically to demonstrate function – then can do mutagenesis of specific sites to see what gets modified and what doesn’t
GABA binding site between alpha and beta subunits (this is why a and b are common subunit types)
BDZ site between alpha and gamma subunits
SEE PHOTO
How is an ionic current of inetrest iosolated in a biological membrane
Two electrode voltage clamp, with ion channel often expressed in Xenopus oocytes
Perfuse with drug/agonist and measure current
Due to oocyte nor expressing any intrinsic ion channles get a very clean current
With one oocyte can do many repeats
allosteric regulators in GABA receptors
DMCM acts as a negative allosteric modulator of GABAa receptors - decreases the effect of GABA at the GABAa receptor
Benzodiazepines (BZDs) chemical structure is the fusion of a benzene ring and a diazepine ring. Benzodiazepines enhance the effect of GABA at the GABAA receptor
Give an example of studying the allosteric regulators on GABAs interaction with GABAa receptors
Campo-Soria 2006
Used a two electrode voltage clamp
Looking at dose response curves for GABA alone, GABA + 1μM of DZP, and GABA + 1μM of DMCM
EC50 (Dose that causes half maximal activation of receptor by agonist) -> In this case in the background concentration of allosteric modulators
EC50 GABA
22 μM in DZP – enhances GABA function
41 μM alone
118 μM in DMCM – More GABA needed to achieve same level of activation of the receptor
Proposed structure of GABAa receptor
Loops A-F participate in ligand binding (seen through mutagenesis studies)
Orthosteric site (ligand binding site)between a and b subunits
C loop of b subunit acts like a clasp to bind GABA
M2 central forming channel pore
Does the C loop have a high or low level of flexibility? whats another name for the C loop of GABAa
High level flexibility
Loop 10
Explain the role of the different loops of GABAa
Movement of loop C is going to create forces to push on the M2-M3 linker opening the ion channel
Loop F (9), Cys loop (7) and Loop 2 are also going to be important, loops have direct contact with the M2-M3 TMD linker
Why are the particular residues facing the lumen of the GABA receptor important
Act as a selectivity filter only allowing Cl- ions through
What important drugs to GABA ion channels bind
volatile anesthetics such as isoflurane or halathane
Also bind propofol which is used in some anasthetic maintinance and some diuretics
Explain the experiment that saw a5IA for enhances cognition
Dawson 2005
Inverse agonist - selective for a5 subunit-containing GABAa receptors called a5IA
Currents recorded in xenopus oocytes
When GABA applied – get current
When inverse agonist applied – less current
also compared to different a subunits and saw this doesnt occur
Explain experiment on a5IA reducing ethanol-induced impairment on word recall
Nutt 2006
Twelve (healthy) male volunteers given α5IA (4 mg) or placebo 2 h prior to being given alcohol in the form of vodka
One-hour later they were asked to memorise a list of 20 words and 0.5 h later (i.e., 4 h after placebo or α5IA administration) were asked to write down as many words from that list as they could recall
Placebo group – only remember about 5 words of the 20
People of had inverse agonist could remember significantly more words (8/9)
Could be used in alzhimers disease but need to be careful you aren’t creating any room for epilepsy type activity in neurones
Also important to consider ethicality of enhancing memory
Feartures of iGluRs
ionotropic glutamate receptors (tetrameric)
Synaptic transmission
AMPA, Kainate (Fast excitation)
NMDA (coincidence detection)
selective cation channels
drug types for iGluRs
Ion channel blockers (memantine - one of the only approved drugs for AD (doesn t work very well), MK-801 – was used as a drug. had nasty side effects so is now used just to study the NMDA receptor)
Allosteric modulators Eg neurosteroids (also act on GABAA receptors)
Disease relevance of iGluRs
Epilepsy
Anxiety
Drug and ethanol abuse
Schizophrenia
Alzheimer’s disease
Pain
Down’s syndrome
Examples of iGluR drugs for disease treatment
Memantine approved for Alzheimer’s disease.
D-serine (NMDA co-agonist) investigated as treatment for Schizophrenia.
Positive allosteric modulators of AMPARs are being investigated for treatment of AD and schizophrenia.
Explain how iGluRs are selective cation channels
Similar concentration of cations in/out the cell. -> Reversal potential for cation channels in neurons is close to 0 mV.
When they open, the cell depolarises. By bringing the cell close to threshold they have an excitatory effect on neurons
Permeability is dictated by ion channel molecule:
NMDARs and some AMPARs allow Ca2+ flux.
Mg2+ blocks NMDARs
Characterisation of iGluRs
Cloning of 18 individual mammalian genes.
mRNA editing and alternative splicing.
Aminoacid sequence alignments.
Ligand binding selectivity for didtinct isoforms of subunits
biochemical analysis of iGluRs
Crystallisation, X-ray-diffraction
Structural studies on soluble domains – particular part of protein cloned and analysed on ots own – this allowed the understanding of the glutamate binding domain of receptors
Overexpression studies allowed study particular role of one or another distinct subunnit
Receptor diversity of glutamate receptors
Ionotropic glutamate receptors - NMDA, AMPA, Kianate
Metabotropic glutamate receptors (groups 1-3)
Features of ionotropic glutamate receptors
ion channels themselves
NMDA, AMPA, Kainate and delta (only by homology but not actually quite a glutamate receptor) - each subytpe has subsets themselves
orphan receptors
Expressed in the Purkinje cells of the cerebellum, inner ear hair cells and hippocampus. Do not bind glutamate (glycine?)
Kainate receptors
- Disease relevance: They have an established role in epileptogenesis (epilepsy). Normal function? Kainate receptors are synoptically activated and critical for the induction of NMDA receptor-independent LTP at the mossy fibre synapses in the hippocampus (have more of a presynaptic role) - may be new drugs to treat epilepsy in the future that are to do with Kinate receptor pharmacology
What is the new nomenclature for ionotropic glutamate receptors
AMPA GluA1-4
NMDA GluN1, GluN2A-D, GluN3A-B
Kainate GluK1-5
Two distinct families of Kainate receptors
GluK1-3
GluK4-5
Explain patch clamps
A phenomenon that occurs when you put clean glass together with a cell membrane
Then take a glass pipette and move it around until they select the sell they want
Manipulate glass pipette to come into contact with this cell
Monitoring electrode resistance (Voltage and current) as this is done
Once suction has been applied you are making a break in the membrane so that you have access to the whole cell - and therefore you are able to see currents of the entire cell
The way that they use this to analyse different receptors is by express in glutamate receptor isoforms in heck cells and then applying glutamate to see if the channel is opened by glutamate
Explain the differentiation of AMPA and NMDA receptors using a patch clamp experiment
fast component through AMPA receptors and the second being a slow component mediated by NMDA receptors
receptors were exposed to glutamate for 1 ms and the AMPA receptors responded quickly and only stayed open for around 1 ms whereas the NMDA receptors stayed open for tens or even hundreds of milliseconds (Depending on the subtype)
AMPA (fast):
τoff < 3ms
NMDA (slow):
τoff < 40 – 2000 ms Depending on subtype
Time constant is the one outcome measure that can be used to describe the characteristic of receptors - it acts as a rate constant for where things are on or off
Explain the common membrane topology of Ionotropic glutamate receptor subunits
Tetramer
Extracellular N-terminus
intracellular C-terminus with a PDZ binding domain that binds to PSD-95
GABA – 5 TMDs, Glutamate – 4TMDs (and 2nd is not a full TMD)
3 transmembrane domains and one reentrant loop
The ligand binding domain is made up from N-terminal regions S1 (N-terminal domain) and S2 (M3-M4 linker) - Regions S1 and S2 form the agonist binding site
Channel gating AMPA
Glutamate binding to one subunit can allow opening of the pore.
Conductance (g = I/V) of AMPA receptors is agonist dependent (due to if more agonist – can bind more (4) subunits and receptor opens more)
Channel gating NMDA
Two molecules of glutamate must bind the GluN2 subunits.
Two molecules of glycine (co-agonist) must bind on the GluN1 subunits. D-serine can also serve this function (instead of glycine)
Conductance of NMDARs is agonist independent (due to all 4 sites needing to be occupied)
Conductance states of AMPA vs NMDA
Multiple (AMPA) vs Single (NMDA) conductance states
Model system for understanding ligand binding domain structure-function relationshops in iGluRs
A construct of isolated S1 and S2 connected by a glycine–threonine linker (S1S2 construct) can be expressed in bacteria and folds into a bilobe structure of the ‘clamshell’ with the upper lobe D1 and lower lobe D2.
Soluble S1S2 construct is capable of binding different iGluR ligands and can be studied using X-ray crystallography and NMR
how does a transmitter open the iGluR channel?
Rest – dimers have interaction – channel closed
Agonist binds – movement in ‘clam shell’ (D1 and D2) - change orientation of residues and separation (still closed – thought to stop excitotoxicity and promote faster transmission)
Agonist bound but desensitized - Chennel opens – D2 separation
^ligand binding domain conformation allow us for distinct conformations of the receptors
So the efficacy of a ligand to activate glutamate receptors depends on its ability to stabalise the maximally closed ligand binding domain of the clamshell conformation
Allosteric modulations have large influences of this change
