Amino Acid NT receptors Flashcards
What are the major excitatory NT receptor in the brain?
iGluRs
What are the three iGluRs? How are they similar? How are they different?
NMDA, AMPA, Kainate
All have similar structures but diverse functions, made of different genes
Tetrameric with 3 distinct domains:
- LBD ligand gating domain: binds receptor
- TMD trans membrane domain: Where signalling happens, 4 subunits come together like a teepee, pore formed by M2 helices
- ATD/NTD animo-terminal domain: Necessary to form complex, help holds 4 subunits together
AMPAR
What is the main role of AMPA receptors? How does it achieve this?
Describe the gating characteristics of AMPARs
Describe the structure of AMPARs
Main role: Depolarize the neuron so NMDARs can be activated
- Rapid activation
- High open probability
- Open/closed states differ by <1 angstrom
- Rapid desensitization
- Subunit dependent recovery
Preferentially heterotetrameric
1 glutamate/subunit to activate (4 total)
AMPAR
What are the AMPAR subunits? Which one is unique and why? What is a result of this?
GluA1/2/3/4
- GluA2 has a single amino acid switch at a critical point*: R (arginine) at narrowest point on pore
- Positively charged R repels positively charged ions (Ca2+. Na+)
- GluA1/3/4 have Q (glutamine) which is neutrally charged, no resistance to cations
*Q/R site, regulates many properties of the receptor
AMPAR
What does the Q/R site in AMPAR subunits regulate? What is the effect on GluA2 containing and GluA2 lacking AMPARs?
- Ca2+ permeability
- GluA2+: Ca2+ impermeable
- GluA2-: Ca2+ permeable
- Polyamine channel block (+ve charged amides)
- GluA2+: insensitive to polyamine block (+ve R repels)
- GluA2-: sensitive to polyamine block (no resistance from Q)
- Single channel conductance (how many ions a single channel can shuttle)
- GluA2+: reduced conductance (R is blocking cations)
- Receptor assembly
- Preferential arrangements depending on Q/R site
AMPAR
Describe a polyamine block and why it would or would not occur
- Polyamines reversibly bind the AMPAR “pocket” when channel is closed
- Glutamate can bind, but the channel will remain closed as long as polyamine is bound
- Polyamines restore rectification
- initially attracted to negative cell
- As voltage increases, less attracted
- Eventually, too positive and polyamine leaves, restoring original function (“rectification”)
Occurs only in AMPAR and KARs that have only Q in the Q/R site (no GluA2)
AMPAR
How do AMPARs assemble? What is the most common subunit configuration?
Individual subunits synthesized in ER, dimerize
Dimers dimerize to form tetramer
Heteromeric dimer formation is more favorable than homomeric dimers
2 Most abundant (and important) receptors in CNS: GluA1/A2 + GluA2/A3
(2x A1:A2 and 2x A2/A3)
AMPAR
What auxiliary subunits to AMPARs complex with?
- TARPs & CNIHs (-ve) attracted to +ve motif in AMPAR
- Traffic AMPAR into synapses, ensure proper destination is reached
- Regulate gating behaviour (fine tuning of signalling)
NMDAR
What is the main role of NMDARs?
Describe NMDARs’ permeability, their gating mechanism and their behaviour
NMDARs act as coincidence detectors
- Premeable to Ca2+ but blocked by Mg2+
- When neuron is depolarized (usually by AMPAR), Mg2+ leaves, allows Ca2+ to enter
- Block is essential for coincidence detection
- Gated by voltage and ligands
- Requires co-activation of L-glutamate and glycine to activate
- 2 glycines and 2 glutamates per receptor
- Usually enough glycine in CSF so [glutamate] is much more important
- Long activations, large conductance, slow-gating behaviour
NMDAR
What are the NMDA subunits? Sub-Sub-units? How are they different?
How do NMDARs assemble? What is a result of this?
GluN1/2/3 | 1 is most stable, 2/3 are variable in terms of funciton/structure)
Each subunit has 4 domains:
- NTD: amino terminal domain
- ABD: agonist binding domain (binds glycine/serine or glutamate)
- TMD: transmembrane domain (ion channel)
- CTD: C terminal domain
Different expression patterns lead to different activities, fine tuning of activities based on regions
NMDAR
How do NMDARs assemble? What is the result of this?
Obligate di- or tri-heteromers
This leads to large receptor diversity
- GluN2A/B/C/D
- GluN3A/B
Vary in terms of:
- Single channel conductance
- Channel maximal open probability
- Sensitivity to glutamate/glycine
- Glutamine deactivation time constant
- Sensitivity to Mg2+ blockade
- Ca2+ permeation
Basically GluN1 + 2A is the fastest, 1 + 2D is the slowest
NMDAR
How many modulators regulate NMDAR activity? What is the takehome?
many many many
Endogenous or exogenous (drugs)
You need to understand how a receptor/protein works at a molecular level if you want to make a good drug
KAR
How are KARs different from NMDARs and AMPARs at a functional level?
What is their function?
It is neuromodulatory instead of hardwired
KARs aren’t fundamental for brain function but make you who you are (govern when you pay attention/handle stress)
Fine-tunes the behaviour of NMDARs and AMPARs
KAR
Describe the ionotropic and metabotropic behaviour of KARs. What makes them unique?
What pathology would benefit from a KAR blocker?
Ionotropic: Summation
e.g. in AMPARs, receptor will open and close for each stimulus. In KARs, very small response per stimulus but slow recovery, which allows for signal summation when stimulus is repeated
Metabotropic: regulates Ca2+ and K+ channel activity
- KARs can couple to voltage gated ion channels making them synchronized, regulating brain function
Blocking KARs in epileptics should prevent seizures
KAR
What genes make up KAR subunits? Are there variations?
Are some unique? How are they organized?
GluK1/2/3/4/5
Splice variants exist
GluK1/2 have Q/R site like AMPARs, controls permeability of channels
Primary subunits (GluK1-3)
- Low affinity subunits
- Required for formation of functional channel
- High homology
Secondary Subunits (GluK4-5)
- High affinity subunits
- modulate receptor properties
- MUST be combined with primary subunits
KAR
What are the auxiliary subunits of KARs? What do they do?
NETO 1/2
- Necessary for signal summation
- Involved in receptor complex trafficking
- Modulate the channel responsiveness/kinetics
PDZ
- Scaffold protein
- PDZ-binding domains found in NETO and all KAR subunits
KAR
What are the neuromodulatory funcitons of KARs?
- Regulate glutamate and GABA release from presynaptic cells
- Increase/decrease either
- Affect intrinsic excitability of cells
- Directly regulating Ca2+ and K+ channel activity
Structural Basis of activation
How does AMPAR gate so quickly?
- Enormous ECD pokes out of synapse, right next to postsynaptic neuron, decreases NT travel time
- Only minor movement required for activation
Structural Basis of activation
Are AMPARs similar to NMDARs in terms of structure or activity?
Structure yes, activity no
structures vary by only a few AA residues, but operate on very different timescales
Structural Basis of activation
What conclusion was made following testing of KAR activation with crosslinking dimers and a cation-binding pocket?
Dimer activation is important for receptor activation but activation itself is promoted by electrostatic interactions between ions and their binding pocks (located in between dimer interfaces)
Structural Basis of activation
Explain Kiss and run mechanisms
Hypothesis: The cation binding pocket is an activation hotspot for all iGluR subtypes
Proof:
- KAR GluK2: has Na+ and Cl- binding pockets
- Cannot open without Na+ binding
- AMPAR GluA2: has a smaller cation binding pocket
- CAN open without Na binding
- BUT Li+ binding (smaller than Na) promotes permanent opening
Conclusion: AMPAR and KAR are structurally identical except for the smaller binding pocket in AMPARs
TAKEHOME: AMPAR and KAR work via kiss and run mechanism, explaining fast gating
NMDAR doesn’t work like this, explaining its slower timescale
Structural Basis of activation
Explain the slow gating mechanism of NMDARs
LBD interfaces are more spread out in NMDARs than in AMPARs and KARs
Top of dimer interface (apex): conserved in iGluRs, doesn’t explain gating behaviour
Bottom of the dimer interface (hinge regions): Y535 is critical for maintenance of channel activation
Mutating this Tyrosine converts NMDAR to AMPAR activity profile (Increases speed of receptor activation/opening)
Slow gating reflects functional coupling between apex and hinge regions
Dynamic synapse
Describe the juvenile plasticity mechanisms involving AMPAR receptors
What iGluR is implicated in the “use it or lose it” principle in synaptic plasticity?
What subunit switch occurs here?
“un-silencing” of synapses
Stimulate neuron at first: no AMPAR response, only NMDAR response
Wait, then stimulate again: AMPAR response detected
- Suggests AMPARs were closeby, not newly synthesised
NMDAR is implicated in use it or lose it
- NMDAR response as well as AMPAR response, strengthens synapse connection
- If two neurons are ‘competing’ for a synapse, the stronger synapse will be favored
Correlated activity promotes GluN2B to switch to GluN2A
Dynamic synapse
Describe a silent synapse
How can this synapse be activated?
How does the amount of silent synapses vary over age? Why?
An exitatory glutamatergic synapse whose post-synaptic membrane contains NMDAR but NO AMPAR (which makes the synapse unresponsive to glutamate)
Synapse remains inactive until it is depolarized via a non-AMPAR mechanism
As the brain matures, silent synapses decrease in number
AMPAR trafficking brings AMPARs to post-synaptic membrane
- Promoted by Ca2+ influx
- Ca binds calmodulin
- Activates CaMKII
- Phosphorylates AMPAR vesicles
- AMPARs inserted in postsynaptic membrane
Dynamic synapse
What describes the maturation of a synapse? What causes this?
How was this figured out?
A redistribution of protein subunits that make up iGluRs/proteins that localise iGluRs
An increase in the ratio of AMPAR:NMDAR
Continued stimulation leads to maturation of the synapse
In early synapses, AMPARs are constitutively traffect into synapses
BUT GluN2B suppresses their insertion via SynGAP
GluN2B -> GluN2A, removes inhibition
Drugs had different pharmacologies in adults vs newborns, different receptors must be at play
Dynamic synapse
What is LTP? Where does it occur? How?
Associated with learning and memory
POST-synaptic change
Hebb’s postulate: neurons that fire together, wire together
Presynaptic LTP:
- Repetitive synaptic activity causes an increase in presynaptic Ca2+, which activates Ca-sensitive Adenylate Cyclase, creates cAMP, causes an increase in glutamate release
- Note: NO CHANGE IN RECEPTOR LEVELS, just more NT is released
NMDA-dependent LTP:
- Responsible for memory circuits
- NMDA - coincidence detector
- Repeated stimulation causes rise in intracellular Ca
- Increased Ca binds Calmodulin, which activates CaMKII
- Autophosphorylated CaMKII phosphorylates AMPARs already present on synaptic membrane
- Increases signal channel conductance
- Open probability goes from 50%->100%
- CaMKII also phosphorylates AMPAR vesicles, more AMPAR on synapse surface
- CaMKII also phosphorylates TARPS which assits with trafficking
Dynamic synapse
What are the three types of LTD seen in class?
NMDAR-dependent LTD
- Some NMDAR firing, some Ca influx into cell
- Activates Protein phosphatase 1 (more sensitive than CaMKII, which doesn’t bind)
- Inhibition of the pathway that inserts AMPARs, AMPAR number decreases
mGluR-dependent LTD
- Activation of metabotropic glutamate receptors leads to endocytosis of AMPAR (unknown process)
eCB-LTD
- Endocannabinoids released by post-synaptic terminal bind to eCB receptors on pre-synaptic side, lowering glutamate release in pre-synaptic neuron (unknown process)
Dynamic synapse
What is the goal of Homeostatic plasticity? How is this achieved?
Goal: keep the plasticity without creating excitotoxicity
Done by scaling down strength of all synaptic inputs while maintaining the ratio of strengths
-
e.g. Neurons A B and C synapse at neuron D
- Each neuron fires with an arbitrary strength of 1
-
A is potentiated to fire more, strength rises to 2
- AD is now twice as strong as BD and CD
-
However, this is too much input for neuron D, so it downscales the strengths of each synapse but not the proportion
- AD = 1.4, BD = 0.7, CD = 0.7
What are the two types of signalling of a GABA receptor?
- Phasic: Similar to iGluR, pulse
- Tonic: Upon release into synapse, GABA diffuses into extrasynaptic space
- Constant activation of delta-containing GABARs (higher affinity)
How are GABARs activated?
Agonists and modulators bind at subunit interfaces
How many subunit families are there? How many of each are there?
What does this lead to?
Which are the most important?
Which is the most common GABA-AR type?
alpha 1-6 subunits
beta 1-4 subunits
gamma 1-3 subunits
delta subunit
epsilon subunit
pi subunit
rho 1-3 subunits
Extreme diversity (many many possible combinations)
alpha1, beta2 and gamma2 are most important
2x alpha + 2x beta + 1 delta/gamma2 is most common type
Describe the structure of GABA-A Rs
Pentamers
4 TM regions, pore formed by TM2
What GABAAR type is found in many brain areas?
A1B2y2 (other combinations have specific localizations)
What is common to all receptors containing the delta subunit?
extrasynaptic receptor
“repellant factor”, says the receptor should not go to synapse
Which subunits confer BDZ sensitivity in GABAARs?
BDZ sensitive: a-1/2/3/5 + b + y2
BDZ insensitive: any receptor with a-4/6
Where do GABAARs synapse? Are these synapses tonic or phasic?
What are their purposes?
Inhibitory synapses (phasic): opposes actions of glutamatergic transmission
Axon initial segment: prevents bidirectional APs from reaching soma
Extrasynaptic receptors (tonic)
Where do neurotransmitters bind GABAARs?
At the INTERFACE*
GABA binds at alpha-beta interfaces, BDZ binds to gamma-alpha interfaces
If a partial and full agonist have the same ability to go from closed to open, how come partial and full agonists don’t elicit the same response?
Because there is a third, flipped state between resting and open states
For a full agonist, there is a net exothermic shift to this state
For a partial agonist, there is a net endothermic shift
There is a net exothermic shift from the flipped state to the open state, so both agonists will open the pore if they can get the receptor into the flipped state
HOWEVER, partial agonists will have more trouble reaching the flipped state
What are 4 ways to strengthen GABAergic transmission?
Phosphorylaton
- Stregthens GABA-AR signalling
Gephyrin and receptor trafficking
- Gephyrin: a scaffolding protein, labels a mature GABAergic synapse
- Similar to PSD-95 in iGluRs
NKCC1 and KCC2
- During development, [Cl] changes
- NKCC1 pumps Cl into cell so that there is an excess of Cl in the cell
- When GABAR opens, cl leaves the cell
- GABA is EXCITATORY here
- KCC2 pumps Cl out of cell so that there is an excess of Cl out of the cell
- When GABAR opens Cl enters cell
- GABA is INHIBITORY here
- NKCC1 and KCC2 are regulated during development so GABA begins as an excitatory NT then becomes Inhibitory later
- Also involved in pain mechanisms (disregulation of pumps)
Neurosteroids
- Allosteric modulation of GABAergic transmission
- Involved in stress
How does LTD affect GABAergic signalling?
- increase in intracellular Ca binds to calmodulin and increases CaMKII
- CaMKII directly phosphorylates GABA-ARs (at the beta/gamma subunit) at specific residues
This has one of two effects on the receptor:
- GABAergic inhibitory postsynaptic currents are prolonged in duration (prolonged hyperpolarization)
- GABAergic IPSCs are increased in amplitude (with unchanged kinetics)
- Due to increased trafficking of receptors to synapse
Could also be affected by calcineurin-driven mechanisms
How is Gephyrin necessary for GABARs?
Scaffold for receptor synapse
Can’t build synapse without scaffold: loss of Gephyrin -> disease
Describe the proposed signalling hub role of Gephyrin
Levels of Gephyrin scaffold increase/decrease depending on the sate of the cell, this effects GABAergic synaptic plasticity
End result is change in strength of GABAergic inhibition
What are the two anions passing through GABAARs? When are each of these depolarizing or hyperpolarizing?
Cl- (depolarizing in early development, hyperpolarizing in mature brain)
HCO3 (always depolarizing)
Describe sensation/pain perception in health and two possible reasons for hypersensitivity to pain
A-b fibres: fast, myelinated, responsible for light touch sensation/fast reflexes
C fibres, slow fibres, responsible for pain sensation
In health: light touch. a-b fibres han inhibit C fibres (via GABA interneurons)
Pain: a-b fibres unable to inhibit C fibre presynaptic termini
touching becomes painful
Two possible mechanisms:
- NKCC1 upregulation -> GABAergic interneuron becomes excitatory
- KCC2 douwnregulation -> unable to hyperpolarize cell
Describe the two potential effects of neurosteroids
Inhibitory neurosteroids
- Act as potent positive allosteric modulators for GABAARs
Excitatory neurosteroids
- Act as potent negative allosteric modulators for GABAARs
- Also act as weak positive allosteric modulators of NMDARs
Other neurosteroids
- Don’t act on GABAAR or NMDAR, affect other cell surface receptors
How are neurosteroids dynamically regulated?
Huge peak during embryonic development, sharp decrease during partruition
Another spike during stress hyporesponsive period
Back to baseline levels into adulthood
What are the two mechanisms that explain why so many mutations lead to epilepsy?
- Mutation affects GABAAR functionality
- Mutation affects trafficking of GABAAR to the cell surface
- (both)
Many different mutations, end result is always epilepsy (hyperactivity)
Name the 4 Endogenous and 2 Exogenous NMDAR blockers/modulators
Endogenous
- Zinc
- NAM
- GluN2A specific
- Protons (H+)
- NAM
- Magnesium
- Voltage dependent pore blocker
- Variable affinity based on GluN2 isoform
- Spermine
- Polyamine
- PAM, selectively enhances GluN2B
Exogenous
- Ifenprodil
- NAM specific to GluN2A
- Channel blockers
- Ketamine, phenyclidine, memantine
- Physical occlusion of pore
*
