Glutamate receptors Flashcards
Which Ca2+-type channels allow calcium influx in the CNS?
N-type channels (CaV2.2)
P/Q-type channels (CaV2.1)
Describe the key steps of neurotransmitter exocytosis
Action potential reaches terminal bouton of presynaptic neuron
–> Triggers voltage-gated Ca2+ channels to open
–> influx of Ca2+ –> cytoskeletal rearrangement –> vesicles fuse with plasma membrane –> neurotransmitter release to synapse –> vesicle recycling
• Only pre-docked vesicles can be exocytosed
By which two types of receptors can neurotransmitters function?
o Ligands
On ligand-gated ion channels
E.g. Glutamate
o GPCRs
Indirect effect on an ion channel
E.g. GABAB¬
Name 5 key ‘fast’ neurotransmitters, the receptors they bind to, and how fast their responses are
o Glutamate iGluRs (kainate, AMPA, NMDA-Rs) mGluRs (group 1,2,3) Excitatory o ACh Via nicotinic receptors o 5HT Via 5HT3 receptors o ATP Via P2XRs o GABA iGABARs (GABAA,C) GABABRs Inhibitory o Glycine iGlyRs Inhibitory o These all trigger responses within a millisecond of binding Response is short-lived
Name 5 of the ‘slower neurotransmitters’ and their receptor type
o Dopamine o Noradrenaline o 5HT o ACh Muscarinic receptors o Neuropeptides o Histamine
Outline the key differences between wired and volume transmission
Wired: Direct synaptic transmission Reuptake via astrocyte Little/no diffusion ∴ limited to activating 1 synapse Equivalent of spraying yourself in the face with a hose Volume: Not as confined as wired transmission Astrocyte reuptake occurs but at a slower rate ∴ diffusion or ‘spillover’ can occur ∴ other neurons can be affected Slower acting/longer effect duration Equivalent to turning on a sprinkler and dancing in the spray
Where are glutamatergic synapses usually found? What else are they known as? What does this allow?
• Usually occur on dendritic spines
o Areas known as the Pre-Synaptic Density (PSD) contain a lot of Glu vesicles
Allows concentrated and direct release
With reference to the key components of a neurotransmitter system, explain what makes glutamate a neurotransmitter
- Requires enzymes to synthesise the neurotransmitter – Glutamate-glutamine shuttle; metabolic processes within presynaptic terminal; glutamate synthase!
- Transporters to get the molecule into cells – Glutamate transporters – Extracellular Amino Acid Transporter (EAAT) 1-5; EAAT2 does 90% of Glu uptake
- Transporters to get the molecule into synaptic vesicles – VGLUT transfers cytosolic Glu to a vesicle; VGLUT1-3; don’t transport Asp
- Receptors that are activated by the molecule – iGluRs, mGluRs; KainateRs, AMPARs, NMDARs
- Requires a way to terminate neurotransmitter action – diffusion, EAAT-mediated uptake
- Glutamate is a non-essential neurotransmitter – you can make all you need.
With the use of a diagram to aid you, show the typical structure of an ionotropic receptor and list the subtypes of glutamate receptor and their unique subunits.
• Ligand-gated ion channel assembly o Channel is inherent to the receptor • Ligand-binding channel opening • 4 subunits, vary between receptors: o 4 transmembrane domains o NMDA: GluN1 GluN2A-D GluN3A,B o AMPA: GluA1-4 o Kainate: GluK1-5
Typical ionotropic structure: https://www.sciencedirect.com/topics/neuroscience/ionotropic-receptors (scroll down)
With the use of a diagram to aid you, show the typical structure of a metabotropic receptor
• G-protein coupled receptors (GPCRs)
o Signal via intracellular G-proteins to second messenger cascades
• Often function as dimers but can work fine as monomers
o 7 transmembrane domains
Typical metabotropic structure: https://www.sciencedirect.com/topics/neuroscience/ionotropic-receptors (scroll down)
List the 3 key features of an AMPA receptor
• Housekeeping receptors of synaptic function
o =/= boring
• Fast transmission, short-lived (normally)
• Na+ influx to cell
With the aid of a diagram, show the subunit topology of an AMPA receptor (including the glutamate binding site) AND explain which subunits in which combinations are required for function
• Tetramer – requires GluA1-4 in any combo
o Homotetramer or heterotetramer
• Q/R site determines the Ca2+ permeability of GluA2
o If have GluA2 as any one of the subunits per receptor, the receptor is then Ca2+ impermeable!
o Normally at least 1 GluA2 subunit per receptor
Subunit topology: https://ars.els-cdn.com/content/image/3-s2.0-B9780123694379500141-f09-01-9780123694379.jpg
List the 4 key properties of an AMPA receptor
• Mediate majority of fast excitatory synaptic transmission (mainly postsynaptic localization) –AMPARs non-selective cation channels permeable to Na+ (goes in) and K+ (goes out) and in some cases Ca2+
• Like all ionotropic glutamate receptors comprised of four subunits to form a tetrameric receptor.
• Four different AMPA receptor subunits in mammals GluA1, GluA2, GluA3 and GluA4 these “mix and match” to produce subtly different receptors.
• AMPA receptors containing GluA2 subunit have very low Ca2+ permeability – due to mRNA editing – positively charged arginine (R) residue expressed instead of neutral glutamine (Q) in pore forming M2 region of GluA2.
o Thus activation of all AMPA receptors leads to an influx of Na+ but these receptors are only permeable to Ca2+ only in the absence of any GluA2(R) subunits (and most AMPA receptors have GluA2(R)).
Explain how RNA editing alters AMPA receptors
RNA editing alters Ca2+ permeability by deaminating adenosine bases in a site-specific manner in double-stranded RNA substrates.
Pharmacology of AMPA receptors: list 2 agonists and antagonists, respectively, and the effects of PAMs
• Agonists: Glu, AMPA (fast desensitisation)
• Antagonists: NBQX (competitive), Telampanel (non-competitive)
• Positive allosteric modulators (PAMs): increase Glu affinity, inhibits Glu-induced fast-desensitisation (cyclothiazide, LY404187)
o AKA Ampakines:
These enhance currents via AMPA receptor channels
Have no effect on their own but enhance the effect of Glu
• Have been in clinical trials for disorders such as Scz, but none have registered for use yet
List 2 facts about Kainate receptors relating to the function and topology, their subunits required for function, and the pores formed during receptor formation
• Not housekeeping
• Similar topology to AMPA
• Subunits:
o GluK1-5
Homotetramers of GluK4/5 do not form functional receptors
o GluK1 and 2 undergo RNA editing at a pore Q/R site; this is regulated in development
• 3 Transmembrane domains (M1, 3 & 4) and 1 re-entrant loop (M2)
• M2 involved in pore formation
• S1 and S2 form ligand binding domain
• Q/R site in M2 domain controls Ca2+ permeability
Pharmacology of kainate receptors: list 2 agonists (not including kainate), and 2 competitive antagonists
- Agonists: kainate, glutamate, domoate
- Antagonists: CNQX, ACET (both competitive antagonists; CNQX also antagonises AMPARs; ACET is selective for GluK1)
- Plant lectin concanavalin A blocks kainate-induced desensitisation
What effects do domoic acid and kainic acid have on animals?
• Domoic acid: o Natural product isolated from marine diatoms from west coast USA & Canada. Responsible for amnesic shellfish poisoning due to consumption of contaminated mussels. o Domoate is a potent AMPA/kainate receptor agonist that can cross the blood brain barrier. o Outcomes include: Loss of short-term memory Motor weakness Seizures Death • Kainic acid seizures in mammals
Explain the roles of kainate receptors and the cells these occur in
• Kainate receptors often found alongside AMPA receptors – need to block AMPA to study Kainate
o Often done with GYKI53655
• KainateRs make excitatory post-synaptic responses
o Confirmed by single-cell patch clamp measurements
o GluK2 is essential for kainate-R mediated EPSPs.
Shown by knockout mice
GluKRs aren’t housekeeping
• CNQX blocks AMPA-Rs and kainate-Rs
• Presynaptic GluKRs can act on the presynaptic neuron
o Can enhance Pr of Glu from mossy fibres
o Also has effects on GABA release
o and has metabotropic effects on neuronal excitability
Cells this occurs in:
Dentate gyrus cells
CA3 pyramidal cells
CA1 pyramidal cells
NMDA receptor structure and function:
• Same structure as AMPA and kainate
• 4 subunits
o Can only arrange as heterotetramers
Obligate heterotetramers due to NR1+2, or NR1+3
o Responsible for excitotoxicity during stroke
Explain the key differences between the 7 subunit genes of glutamate receptors
GluN1 – required
• If you KO the GluN1 gene, no NMDARs can form
• Binds glycine – required for NMDAR function!
• Only 1 gene but 8 spliced forms
• Expressed everywhere in the brain
GluN2A-D – requires at least 1
• Binds Glu
• These genes are differentially expressed in different brain areas and at different ages
• Little A at birth, increases with age
• Always B
• C doesn’t show up until cerebellum develops – largely localised to the cerebellum
• D is lost over time
GluN3A-B
• Comparably little known about these.
List 3 important properties of NMDA receptors and explain how these relate to function:
- High Ca2+ permeability – always, unlike with GluA2
- Mg2+ blocks the channel at negative (resting) Vmem
a. This is voltage-dependent:
i. In order for Mg2+ block of NMDAR to occur the channel must be open - ∴ glycine and glutamate must be bound to their binding sites on the NMDA receptor.
ii. As membrane potential is depolarised, the Mg2+ block of the NMDA receptor channel is progressively removed.
iii. AMPARs often present at same synapse as NMDARs. Activation of AMPARs depolarises the membrane sufficiently to remove Mg2+ block of NMDARs.
iv. Thus Ca2+ entry through NMDARs is dependent on pre- and postsynaptic elements being active at the same time i.e. the NMDAR is a coincidence detector.
v. By ~-35 mV, the Mg2+ block of the NMDA receptor channel is mostly removed ∴ inward current through the NMDAR is voltage dependent and transmitter gated
vi. Mg2+ presence doesn’t lead to a slower response in NMDARs; slow responses are inherent to NMDARs. Slow responses continue in the absence of Mg2+ - Glycine is a necessary co-agonist –> obligate heterotetramer
Explain the synaptic physiology of NMDARs
• NMDARs mediate slowly rising, long-lasting EPSPs via Na+ and Ca2+ influx through the channel – NOT BECAUSE OF Mg2+
o Studies in absence of Mg2+ have shown this
• Subunit composition affects the time of the EPSP decay
o Glu isn’t in the synapse for any more than 5ms
Just diffuses away very quickly
• Ca2+ influx –> activation of enzymes; regulation of ion channel opening; affects gene expression
o Can also –>
Synaptic plasticity – ability to change strength of synaptic connections, and consolidate new pathways. NMDARs are specifically involved in:
• Long-term potentiation (LTP) – long-lasting potentiation of synaptic transmission
• Long-term depression (LTD) – long-lasting depression of synaptic transmission
• NMDAR antagonists block these processes (e.g. AP5)
Molecular pharmacology and structure of NMDARs - overview:
- Agonists and antagonists for every binding site on the molecule
- Pore blockers exist, bind the pore and block it
- can study the receptor through mutagenesis – KOs etc
Molecular pharmacology and structure of NMDARs - Glu site agonists:
o Glu o NMDA (N-methyl-D-aspartate)
Molecular pharmacology and structure of NMDARs - Glu site competitive antagonists:
AP5 • Glu analogue • Widely used as a blocker for NMDA • Been around for years • Can’t cross BBB CPPene • Crosses BBB, can be dosed and enter brain
Molecular pharmacology and structure of NMDARs - Gly site agonists:
o Gly
Affinity of glycine for GluN1 depends on type of GluN2 subunit in tetramer
o D-serine
D-serine is released by astrocytes and may act as a transmitter in some areas of the CNS
Molecular pharmacology and structure of NMDARs - Gly site antagonists:
o Kynurenate – an endogenous compound
produced by glial cells. Antagonist at
the glycine binding site
Many potent and selective antagonists such as 5,7-dichlorokynurenate (5,7-DCKN) have been developed by medicinal chemists in the search for therapeutic agents.
Molecular pharmacology and structure of NMDARs - N-terminal domain:
o Eliprodil – selectively binds to the dimer interface between N-terminal domains of the GluN1 and GluN2B subunits leading to inhibition of the NMDA receptor
is a negative allosteric modulator (binds to a site other than the glutamate/glycine binding sites).
Channel blockers (uncompetitive antagonists) - low affinity:
Ketamine (Ketalar): dissociative general anaesthetic; induces a state of sedation, immobility and analgesia. Used in veterinary practice and also licensed for use in humans in short-term treatment of pain resulting from cancer, peripheral nerve disease or spinal cord injury. “Special K” – drug of abuse
Memantine: well tolerated low affinity channel blocker used in the clinic for the treatment of memory impairment in moderate-severe Alzheimer’s disease, vascular dementia and the dementia of Parkinson’s disease.
Channel blockers (uncompetitive antagonists) - high affinity:
Phencyclidine (PCP): once used as general anaesthetic withdrawn due to hallucinogenic effects. Now a commonly abused drug (street name - angel dust).
MK-801 (dizocilpine): high affinity channel blocker, withdrawn from clinical trials for stroke due to adverse side effects such as hallucinations and memory impairment.
List 4 pathophysiological roles of NMDA receptors:
- Excitotoxicity (through excessive entry of Ca2+) –> neuronal cell death (e.g. in cerebral ischaemia, Alzheimer’s and Parkinson’s disease).
- Epilepsy – NMDARs involved in development and expression of seizures. Anticonvulsant activity of NMDAR antagonists correlates with their affinity for the NMDAR.
- Transmission of pain responses - NMDARs expressed on sensory neurones. NMDAR-dependent maladaptive plasticity (e.g. wind-up) in neuronal pain pathways (hyperalgesia) can be blocked with antagonists.
- Schizophrenia is associated with NMDAR hypofunction e.g. PCP causes psychotic episodes that resemble schizophrenia.
List 4 facts about GluN3 subunits:
• Excitatory ionotropic Glycine Receptor
• Doesn’t require Glu binding to activate the receptor and have the response
• Not blocked by strychnine
o D-serine is a partial agonist
• Gly inhibits NR1 but activates NR3, but we still don’t know how these work
o If you block NR1, NR3 works better
o Redox modulation may have some effect on this
Explain what kind of receptors metabotropic glutamate receptors are, and list the subfamilies of mGluRs
• GPCRs – Glu-activated • 8 in mammals; 3 subfamilies o mGluR1-8 o subfamilies: Group 1: • mGluR1, 5 • signal via Gq • agonised by DHPG Group 2: • mGlu2+3 • signal via Gi/o o inhibitory • agonised by LY404039 Group 3: • mGlu4,6-8 • signal via Gi/o o inhibitory • agonised by L-AP4
Which signalling pathway are mGlu group 1 Rs coupled to? What are the intracellular effects of this pathway?
Group 1s are coupled via Gq to PLC:
Glu/DHPG binding –> αq is given GTP–> PLCβ (GTP–>GDP, αq dephosphorylated)
–> PIP2 –> IP3 + DAG
IP3 –> IP3R (on ER) Ca2+ released from ER into cell, increases i[Ca2+] –> Ca2+-dependent processes
DAG + Ca2+ activate PKC –> protein phosphorylation
Which signalling pathway are mGlu group 2+3 Rs coupled to? What are the intracellular effects of this pathway?
Group 2 + 3 are negatively coupled to adenylyl cyclase:
LY404039/L-AP4 –> αi/o phosphorylated; inhibited adenylyl cyclase –> decreased ATP–>cAMP decreases
βγ complex activated G-protein coupled Inwardly Rectifying K+ channels (GIRKs) + inhibits VDCCs
With the aid of a diagram, describe typical mGluR structure, showing drug binding sites:
• Bi-lobed N-terminal extracellular domain; contains Glu binding site (orthosteric) – can bind 2 Glu molecules if needed
• Cysteine-rich domain – involved in maintaining 3° structure
• 7 transmembrane domains (TMDs) – shared with other GPCR families
o Allosteric modulators bind here
• 2nd intracellular loop
o Involved in G-protein coupling
o Determines transduction mechanism
Typical structure: https://www.abcam.com/content/metabotropic-glutamate-receptors (scroll down)
Is mGluR1 hetero- or homodimeric? Explain its structure and relate this to function
bridge between Cys residues present on Ligand-Binding site 1 of the Ligand Binding Regions
o Bilobed structures (LB1+2) of each protomer are flexible – can form open/closed conformations
o X-ray structure showed Glu bound to both protomers:
1 with lobes closed
1 with lobes open
Corresponds to activated state
o Research suggests lobes should be closed in both protomers to achieve the fully activated state
o Spontaneous lobe closure in agonist absence mGluR activation (constitutive activity) & basal activity of receptor – e.g. basal levels of phosphoinositide hydrolysis
Blocked by inverse agonists
Glu binding stabilises activate state; orthosteric antagonists bind to the resting state – prevents lobe closure which would lead to receptor activating
What is MPEP and what does it do?
MPEP is a negative allosteric modulator of mGlu5:
Increasing [MPEP] decreased max response of a [Gu] response curve on rat mGlu5
MPEP binds to a site in the TM region of mGlu5 to inhibit the Glu-stimulated rise in i[Ca2+]
MPEP but not MCPG acts as an inverse agonist to block constitutive activity of rat mGlu5a:
MPEP blocks basal production of inositol phosphates (IP) (occurs through constitutive activation of mGlu5a) – control shows IP production in cells not expressing mGlu5a
MPEP but not orthosteric antagonist (MCPG) blocks basal IP production
∴ MPEP is a NAM and inverse agonist of mGlu5.
What is Ro01-6128?
A positive allosteric modulator of mGlu1:
o Conc.-response curves for effect of (S)-DHPG on mGlu1 are shifted to the left in presence of Ro01-6128
o 5-fold decrease in EC50 value, and 1.2-fold increase in maximum response as observed in the presence of Ro01-6128 = a change in the efficacy of orthosteric agonist DHPG.
List the general properties of mGluRs:
- Depending on subtype can be pre- and/or post-synaptically localised.
- Generally play a modulatory role in synaptic transmission.
- Postsynaptic group I mGlu receptors mediate slow depolarization (EPSP)
- Presynaptic group II and III mGlu receptors decrease neurotransmitter release.
- Involved in the modulation of signalling through K+ and Ca2+– control excitability of neurones.
- Metabotropic glutamate receptors were desirable targets for drug discovery.
- mGluR activation is also involved in various forms of synaptic plasticity
- This include short term plasticity at a variety of synapses
- Also reported involvement in forms of LTP and in particular LTD
- A few minutes’ application of the group-1 agonist DHPG will induce hippocampal LTD
