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Pharmacology of the nervous system > Metabotropic receptors: structure & pharmacology > Flashcards

Metabotropic receptors: structure & pharmacology Flashcards

1
Q

What are group 1 mGluRs? (subtypes, signalling mechanisms & agonists)

A

mGlu1 + mGlu5

Gq signalling: activate PLC → produce DIP3 etc → increase intracellular calcium

Agonist = DHPG

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2
Q

What are group II mGluRs? (subtypes, signalling mechanisms & agonists)

A

mGlu2 + mGlu3

Gi/Go signalling: inhibit adenylyl cyclase → decrease cAMP

Agonist = LY404039

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3
Q

What are group III mGluRs? (subtypes, signalling mechanisms & agonists)

A

mGlu4, mGlu6, mGlu7 mGlu8

Agonist = (S)-AP4

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4
Q

What is the grouping of mGluRs based on?

A

Placed into three groups according to amino acid sequence homology, signal transduction mechanism and agonist pharmacology

*Signal transduction is that observed when receptor expressed in non-neuronal cell line (e.g. HEK cell, chinese hamster)- in neurons alternative coupling may be observed e.g. to ion channels

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5
Q

What is the role of Group I mGluRs?

A

Postsynaptic: mediate slow depolarisation

*single stimulus & measure single AP- don’t get postsynaptic response via group I, but if give trains of stimulation but can sometimes pick up very slow excitatory response in some brain regions (Steve skeptical)

Affect excitability post-synaptically by modulating AMPARs + NMDARs, and modulating ion channels

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6
Q

What is the role of Group II + III mGluRs?

A

Presynaptic: decrease glutamate release (autoreceptors) + GABA release (heteroreceptors expressed on GABA / other transmitter terminals)

  • Reduce intracellular calcium
  • modulation of signalling through K⁺ and Ca²⁺ channels as well as iGluRs- control excitability of neurons (therefore desirable drug targets but none on market yet)

Group II may sometimes be expressed post-synaptically but controversial (some receptors found on glial cells)

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7
Q

Outline the topology of mGluRs

A

Venus fly trap domain (VFD), within the ATD contains glutamate binding site (Class C GPCRs therefore ligand binding is on extracellular ATD)

LINKER region with CYSTEINE residues joins VFD + TMD

7 transmembrane domains (7-TMD) in common with other GPCR families (allosteric modulators bind at sites in the TMD)

2nd & 3rd intracellular loop involved in G-protein coupling + determining transduction mechanism

C-terminal tail domain (internal, long): subject to ALTERNATIVE SPLICING, regulation by PHOSPHORYLATION and protein-protein interactions (properties vary: e.g. mGlu5 has C-terminal versions A & B, can affect signalling / trafficking)

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8
Q

What has X-ray crystallography revealed about mGlu1?

LBR of mGlu1 has been expressed in soluble form + crystallised

A

mGlu1 is HOMODIMERIC: 2 mGlu1 subunits (protomers) each with LB1 + LB2 - can have 1 or 2 glutamate molecules bound

Protomers joined by a DISULPHIDE BRIDGE between cysteine residues present on the LB1 regions

Bilobed structures (LB1+LB2) of each promoter are flexible and can form open or closed conformations

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9
Q

What has X-ray crystallography revealed about glutamate binding to mGlu1?

A

Glu bound to both protomers, one with lobes open, one with lobes closed - corresponds to an activated state

Evidence suggests that lobes should be closed with glutamate bound in both protomers for FULLY activated state (one closed = partially activated)

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10
Q

Why do splice variants of mGlu1 differ?

A

For some variants: spontaneous lobe closure (in absence of orthosteric agonist) can lead to mGluR activation (constitutive activity - partial activation) + basal activity of the receptor

e. g. basal levels of phosphoinositide hydrolysis - blocked by inverse agonists
* lobe closure without agonist is not favourable - more activation will occur if glutamate is present

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11
Q

How does glutamate affect the activation of mGluRs?

A

Glutamate binding stabilises the activated state (lobes closed)

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12
Q

How do orthosteric antagonists affect mGluRs? Example?

A

Bind to the resting state to prevent the lobe closure that would lead to receptor activation

MCPG = an orthosteric antagonist i.e. binds to LBD (wedges lobe to stop it closing)
*MCPG doesn’t distinguish between Groups I/II/III

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13
Q

What is MPEP?

A

NAM of mGlu5 (acts like an inverse agonist)

Binds amino acid residues in TMIII(3) + TMVII(7)
- may stop VFD closing but definitely stops G-protein activation in TMD + therefore inhibits the Glu-stimulated rise in [Ca]i

Drugs like NAMs affect constitutive activity: prevents activation of the receptors

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14
Q

How does MPEP affect the glutamate conc-response curve?

A
  • ↑MPEP reduces the glutamate response
    • doesn’t shift conc-response curve to the right like you would with a competitive antagonist, but do REDUCE THE MAX RESPONSE (typical of non-competitive antagonist)
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15
Q

What is an inverse agonist and what is a neutral antagonist?

A

Receptor must have constitutive (basal) activity level in absence of ligand in order for inverse agonist to work

Agonist increases the activity of a receptor above its basal level, inverse agonist decreases the activity below the basal level.

A neutral antagonist has no effect in the absence of an agonist or inverse agonist- but can block the effect of both

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16
Q

What did Pagano et al (2000) show about MPEP and MCPG?

A

MPEP acts as inverse agonist to block constitutive activity of rat mGlu5a (Orthosteric antagonist MCPG does not).

Expressed control cells (no mGlu5), then mGlu5a cells with control, Glu, MPEP or MCPG: measured IP3 turnover

  1. Control has low IP3 turnover - therefore mGlu5 increases basal activity without adding agonist
  2. MPEP blocks basal production of inositol phosphates (IP), therefore blocks constitutive activation of mGlu5a
  3. MCPG does not block constitutive activation (same as mGlu5 cells with nothing added)
  4. Glutamate = bigger increase in basal activity

Shows that constitutive activity is independent of glutamate binding (as not affected by MCPG)

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17
Q

What is the X-ray crystal structure of mGlu1 TM REGION show about structure?

A

Parallel dimer formed by 7TM of mGlu1: interface mediated by several cholesterol molecules

*Cholesterol links the 2 TM regions, stabilises dimer

Interactions in both VFD (disulphide bridge) and TMD (cholesterol links) may be important for dimer formation

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18
Q

What does X-ray crystallography of mGlu1 selective NAM bound to TM show?

A

NARROW mGlu1 NAM binding site

Formed by extracellular loop 2 (ECL2) and TM helices II, III, V, VI and VII (largely hydrophobic binding site: only 1 H-bond between ligand and Thr815)

This site partially overlaps with ORTHOSTERIC sites of Class A GPCRs but is more restricted

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19
Q

What is is ionic lock?

A

Occurs in class A GPCRs

Mediated by a salt bridge between an ARGININE residue on HELIX III(3) and an ACIDIC residue on HELIX VI(6), leading to stabilisation of the inactive state by restricted outward movement of helix VI which is required for activation

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20
Q

What is the mechanism of an mGlu1 selective NAM?

A

With mGlu1 NAM: intracellular side of TMD adopts INACTIVE conformation - stabilised by a SALT BRIDGE between a LYSINE (K678) and GLUTAMATE (E783) residue on helices III(3) and VI(6), respectively

Similar to ionic lock in Class A GPCRs responsible for inactive state observed upon NAM binding to mGlu1

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21
Q

What evidence suggests mGluRs may be heterodimers?

A

Crystals initially suggests they were homodimers (one subunit) but recent studies using time resolved (tr-FRET) to analyse cell surface receptors expressed in cell lines have shown that heterodimers may also exist

DOUMAZANE ET AL (2011) - combined trFRET with CLIP-and SNAP-tagged rat mGluR subunits expressed in mammalian cell lines (clip/snap = fluorescent tags)

  • Heterodimers formed by subtypes that couple to the same G protein - even mGlu2 + mGlu4 (intergroup dimers with same signalling mechanism)
  • Also showed that only 11/21 possible pairs formed (mGlu6 not included)
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22
Q

What is significant about the 5-HT2A receptor in terms of mGluRs?

A

Delille et al (2012): mGlu2 + 5-HT₂A receptors shown to form heterocomplexes in cell membrane (also used CLIP/SNAP tagging)

  • Investigations ongoing: are these heterodimers between receptor families, or tightly associated heterocomplexes?
  • Delille’s results suggest the heterocomplexes do not translate into second messenger effects (didn’t make a difference to effects of 5-HT2a/mGlu2 agonists etc.). Although there is well-documented functional cross-talk of the two receptors in the brain, these results challenge the biological relevance of the 5-HT2A-mGlu2 heterocomplex (i.e. receptors definitely interact, but heterocomplex itself may not be relevant)
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23
Q

What is the limitations of studies showing mGluR heterodimers?

A

Although mGlu subtypes been shown to co-localise in some neurons - are heterodimers expressed in the CNS?

  • studies like Doumazane (2011) showing that they interact is in cell lines that are artificially manipulated to express subunits (labelled the subunits then transfected them into cells)
  • difficult to show in neurons - know that a population of neurons can express different populations of mGluRs but proving that they link together is difficult
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24
Q

What is significant about PHCC? What does this tell us about application of research?

A

Conn et al (2014): expressed mGlu4 alone or in combination with mGlu2, in non-neuronal cell line

PHCC potentiates responses (CRC shifted left) in mGlu4 homomers (mGlu4 PAM) - but mGlu2/4 heterodimers are insensitive to PHCCC

PHCC potentiates glutamate responses in some native systems where mGlu4 expression occurs in absence of mGlu2 - but may have a drug that works on cell line but then in vivo if mGlu4 always associated with something else, drug may not work !!!

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25
Q

What is significant about VU0155041 (mGlu4 PAM)?

A

Conn et al (2014): potentiates responses to glutamate or mGlu2 agonists on mGlu2/4 heteromers (CRC shifts left)

(unlike PHCCC)

*Suggests transactivation between two sides of mGlu2/4 dimer is possible - agonist binds mGlu2 + potentiator binds mGlu4; still get shift of CRC; functional connectivity between two subunits, don’t need to activate the same subunit that the potentiator is working on!

Therefore some PAMs may potentiate responses to mGlu homodimers only, whereas others will also potentiate heterodimers (depends on structure of the PAM)

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26
Q

Why do PAMs and NAMs tend to be more subtype selective?

A

They bind diff regions to orthosteric drugs + there is more AA variation at these regions (where as VFD tend to be more similar across subtypes)

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27
Q

What are the advantages of using mGluR allosteric modulators as therapeutics?

A

Can fine tune receptor activity (modulating responses rather than sledgehammer effect, less side effects)

Can achieved high mGluR subtype selectivity (also avoiding side effects) - more difficult with orthosteric drugs due to conservation of LBD between subunits

Usually non-polar (orthosteric drugs tend to be polar AA) therefore more ‘drug like’ + greater potential to cross BBB

Only effective when glutamate is present

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28
Q

What are the disadvantages of using mGluR allosteric modulators as therapeutics?

A

Activity on native receptors (brain) does not always correlate with effect in recombinant receptor assays in cell lines - due to ligand-biased signalling behaviour (functional selectivity), differences in receptor expression levels and coupling efficiencies to effector mechanisms

Allosteric modulatory activity depends on receptor expression levels and efficiency of coupling to effector mechanism

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29
Q

What are Ago-PAMs?

A

PAMs that have intrinsic allosteric agonist activity

*don’t just potentiate the effects of orthosteric agonists, can actually activate the receptor as well

Ago-PAM activity depends on receptor expression level and/or coupling efficiency to the second messenger system that is being assayed

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30
Q

What are examples of how PAMs and ago-PAMs are effected by receptor expression levels, and coupling efficiency in native systems?

A

Ca²⁺ mobilisation assays: some mGlu5 PAMs showed AGO-PAM activity if cells had HIGH (not low) mGlu5 EXPRESSION (i.e. only agonist activity if high mGlu5 expression)
- when tested in electrophysiological assays in NATIVE systems where mGlu5 expression is LOW - ONLY get POTENTIATOR activity, not agonist activity)

i. e. Some ago-PAMs have only demonstrated PAM activity in native system, because native tissue has lower receptor expression than in cell lines (assays used high expression as easier to pick up signals)
* Must consider impact of receptor expression levels and coupling efficiency in native systems when developing PAMs

31
Q

What kind of mGluR drugs are being developed for schizophrenia? Why?

A

mGlu5 PAMs

  • greater therapeutic than ago-PAMs for treatment of COGNITIVE deficits (not tackled by current drugs)
  • can enhance both hippocampal LTP and LTD while preserving the normal patterns of presynaptic activity that are needed to induce these forms of plasticity and enhance cognitive function in animal models
  • However, mGlu5 ago-PAMs and orthosteric agonists shift the balance in favour of enhancing LTD over LTP and have been shown to induce seizure activity in animal models - mGlu5 ago-PAMs should be avoided
32
Q

Where do orthosteric mGluRs bind / how do they affect responses?

A

Orthosteric agonist: same site as (S)-glutamate (in VFD), activates receptor

Orthosteric competitive antagonist: competes for same site as Glu but no effect on its own - inhibits agonist activity (shifts dose response curve of Glu to the RIGHT)

33
Q

Where do mGluRs NAMs + PAMs bind / how do they affect responses?

A

NAM: different site to (S)-glutamate (usually in 7-TMD region) and inhibits effect of orthosteric agonists

PAM: different site to (S)-glutamate (usually in 7-TMD region) + increases potency of orthosteric agonist (shifts dose-response Glu curve LEFT)
- Some may also increase MAX response (i.e. increase the efficacy of orthosteric agonist)

*Note PAMs and NAMs have no effect on their own

Ago-PAM: PAMs that also have intrinsic allosteric agonist activity in the absence of an orthosteric agonist

34
Q

Where do mGluR allosteric and inverse agonists bind? How do they affect responses?

A

Allosteric agonist: binds 7-TMD region + activates receptor (no potentiator activity)

Inverse agonist: inhibits constitutive activity (i.e. basal activity) *some NAMS will also do this e.g. MPEP

35
Q

mGluRs can couple to range of effector mechanisms..

A

might not bother with this card..

36
Q

What determines the coupling mechanism / downstream effects of mGluRs?

A

Their synaptic localisation

Group I solely postsynaptic (mGlu1 + mGlu5 never seen on presynaptic terminals)

37
Q

Which mGluRs are present in the presynaptic active zone? What are their properties?

A

Only mGlu7 receptors present in presynaptic ACTIVE zone

LOW AFFINITY for glutamate (Ki 900 µm), so conc. has to be high, but as it is situated right where glutamate is released may reach ~1mM therefore possible to activate

Serves as an autoreceptor - detects glutamate present at high concentrations and stops further build up (probably only mGluR that responds to SINGLE vesicle release of glutamate + can then decrease further release of glutamate as single AP reaches synapse)

mGlu4/mGlu8 (other group III) are expressed away from active zone

38
Q

Where are group II + III mGluRs located?

What are the exceptions?

A

Group II & III typically presynaptic: mGlu2/3/4/8 will only be activated by HIGH frequency repetitive synaptic stimulation as glutamate must diffuse to reach them (away from site of transmitter release)

BUT group II (mGlu2 + mGlu3) are ALSO postsynaptic (note mGlu6 generally ignored as only expressed in retina)

39
Q

What is the most common coupling mechanism of group I mGluRs?

A

Can modulate range of intracellular signalling pathways by coupling to variety of GPCRs in both recombinant systems and neurons

Most common = typical canonical signalling: Gq/11 → PLC → IP3 → Ca²⁺ (IP3 binds receptors in endoplasmic reticulum → calcium release)

PLC also produces DAG → PKC activated → phosphorylation

PKC can activate PLD and PLA₂ and modulate a variety of ion channels

40
Q

What is an additional coupling mechanism of Group I mGluRs (apart from Gq/11)

A

Can also couple to Gαs: (AC → increased cAMP production → PKA activation)

  • mGlu1a also couples to Gαs via G5
  • mGlu1a can couple to Gi/o (inhibit ACs)
  • This can modulate MAPK (activate ERK2) + ion channels (e.g. can negatively modulate P/Q + N-type Ca²⁺channels via GβƔ-subunits of Gi/o).

MAPKs also activated by PKC; therefore mGlu1a can activate MAPKs via Gq OR via Gi/o

MAPKs phosphorylate things + change gene transcription (downstream changes to nucleus)

41
Q

What did Sheffler and Conn (2008) show with allosteric modulators of mGlu1a? (calcium/cAMP)

A

BHK cells (kidney): loaded with fluorescent Ca indicator: glutamate conc-response curves in presence of fixed conc of 3 PAMs (company: Rosh)

Assays with Group I receptors show STEEP response to glutamate

  1. 1µM of PAMs: left CRC shift: typical Gq signalling
  2. 500nM PAMs: cAMP assay (to detect Gs signalling): left CRC shift again, but increases responses even at very low Glu concentrations - increases BASELINE response
    * one compound seems to be an Ago-PAM: although all 3 increases baseline a bit, only one increases it significantly and therefore has Ago-PAM effects (other 2 PAM only)

Conclusion: Stimulation of Gq (calcium increase) = PAM effects (no baseline increase). Stimulation of Gs (cAMP increase) = agonist effect (Ago-PAM effects)

42
Q

What did Sheffler and Conn (2008) show with allosteric modulators of mGlu1a? (MAPK effects)

A

MAPK = family of protein kinases; ERK is one that couples with Group I mGluRs (gets phosphorylated when activated)

Assay: measuring phosphorylated ERK - could be by Gi/o proteins or could be 2ndary PKC activation (Gq)

Increasing concentrations of all 3 ROSH mGlu1a PAMs WITHOUT adding glutamate shows ERK phosphorylation: therefore allosteric AGONIST effects

43
Q

Overall; what did Sheffler and Conn’s (2008) findings tell us?

A

mGlu1a have 3 different signalling mechanisms which may be via different G proteins - slightly different effects of the different PAM compounds on 3 different assays

Compounds can act be pure potentiators, as mixed potentiator agonists (Ago-PAMs) or as pure agonists

ERK phosphorylation assay = pure agonists
cAMP assay (Gs) = one ago-PAM, two PAMs
calcium assay (Gq) = PAM only

The compounds exhibit FUNCTIONAL SELECTIVITY - so one compound won’t alway act as potentiator/agonist -
it depends on which signalling pathway you are looking at

44
Q

Explain how functional selectivity works and the implications of this

A

*Allosteric modulators can fine tune ability of agonists to STABILISE SPECIFIC ACTIVE CONFORMATIONS and thereby differentially alter effects of Glu on SPECIFIC SIGNALLING PATHWAYS

Implication: can attempt to develop compounds that regulate specific signal transduction pathways involved in CNS disorders but not in physiological brain functions

e.g. one disease may cause decrease in ERK activation - find compounds that just boost ERK activity of receptors without affecting other functions that are not involved in the diseases (no mGluR agonist/PAMs/NAMs licensed, must most companies have investigated this at some points)

45
Q

What is ligand biased signalling?

A

Different orthosteric agonists activate different signalling pathways depending on how they interact with LBD (diff amino acids)

*this method can be used to find new compounds that only activate certain pathways

46
Q

How did Emery et al (2012) demonstrate ligand biased signalling?

A

Cloned Chinese hamster Ova cells + expressed mGlu1

  • Applied range of orthosteric agonists (Glu, aspartate - endogenous) + (quis / L-CA / DHPG - synthetics). Also applied mGlu1-specific allosteric antagonist (YM..) - control measure to show effects are due to mGlu1 activation
    1. All orthosteric antagonists increased PI hydrolysis (therefore all activated Gq pathway - completely inhibited by the allosteric antagonist)
    2. All increased transient pERK (blocked by antagonist) - increased 5 mins after adding agonist
    3. Some (Glu + asparatate) increased DELAYED pERK (i.e. ERK activation 24 hours after agonist) - L-CA less so + Quis. & DHPG no sustained activation

4) Viability (% of control) = cell survival (control, starve serum 3 days and get ~35% viability/living cells). Treat with agonist during 3 day period, some mGlu1 agonists show conc-dependent stimulation of cell survival
- viability increased only with Glu, aspartate and L-CA (3/5)

Conc: PI hydrolysis with all compounds, transient pERK with all compounds, sustained pERK - only some agonists have good effects, cell survival - only some have good effects

47
Q

How did Emery et al (2012) demonstrate the role of delayed ERK activation?

A

SUSTAINED pERK is contributing to cell survival!!

Cell survival promoted by certain mGlu1 agonists through ligand biased signalling

48
Q

How did Emery et al (2012) demonstrate the role of mGlu1 mutations in cell signalling?

A

Control cell lines: expressed no receptor (transfect with empty vector) or express normal mGlu1

Threonine 188 mutation to alanine (T188A): abolishes PI hydrolysis + transient pERK, but no effect on sustained pERK or cell survival.

*sustained pERK does not involved T188; involves binding to arginine 323 and glycine 409 - occurs via beta arrestin signalling rather than G-proteins

Identified series of AAs in glutamate binding domain interaction with different AAs within this binding domain is important for different types of signalling

49
Q

What are examples of G protein biased + unbiased agonists (Emery et al 2012)?

A

Biased = quisqualate & DPGG: (only activate the PLC signalling pathway - Gq biased)

Unbiased = Glu, aspartate, cysteate (L-CA): can activate PLC pathway but also another pathway that leads to sustained pERK

50
Q

What are beta arrestins?

A

Important for regulating GPCRs and can often cause desensitisation of GPCRs

Can also stimulate various signalling cascades including sustained pERK (transient activation is via G proteins)

51
Q

What are βArrestin biased agonists?

A

Glutarate, succinate

Activate B-arrestin pathway but cannot activate the PLC pathway - interact with the arginine/glycine amino acids but not with threonine 188

52
Q

What are homers (structure/attachments)

A

Several types exist: Homer 1a/b/c Homer 2 & Homer 3

Proteins with an N-terminus (target binding domain) that interacts with the C-termini of mGlu1a or mGlu5

Homer C-terminus contains coiled-coil domain: this can interact with other coiled-coil domains to form chains (allows Homer dimerisation + interaction of mGlu1 or mGlu5 + another protein e.g. IP3 receptor via the free target domain in the homer dimer)

53
Q

How does Homer affect IP3 mechanisms?

A

*Homer 1b, 1c, 2 or 3 can attach mGlu1a or mGlu5 receptors to the IP3 receptor, leading to more efficient coupling of receptor to intracellular Ca²⁺ release

Keeps IP3 receptor in local vicinity so that when PLC activated & IP3 increased - stimulates release of calcium from endoplasmic reticulum because IP3 receptors located close to the mGluRs

This mechanism may also be involved in synaptic development & plasticity

54
Q

How has the role of Homers been demonstrated experimentally?

A

If remove / KO homer: will get less calcium release (as IP3 moving away from mGlu1/mGlu5)

55
Q

What is the role of Homer 1a?

A

It has no coiled coil domain + can compete with Homer 1b/ 1c/2/3 for binding to mGluRs or IP3 receptors

*Increasing Homer 1a expression prevents mGlu-IP3 coupling. Over-excitation of neurons leads to high expression - may be a mechanism to control Ca²⁺ release e.g. NMDA treatment → excitotoxicity can increase Homer 1a expression, minimising the intracellular calcium release

Chronic stress can also increase Homer 1a expression (minimises over activation of neurons, neuroprotective)

This mechanism may also be involved in synaptic development & plasticity

56
Q

What ion channels can mGluRs couple to?

A

Group I

Negatively modulate K+ channels (leak conductance or IK:AHP)

Positively modulate NMDARs

Group II / III

Negatively modulate Ca channels

Positively modulate GIRK (G protein-coupled inwardly rectifying potassium channel)

57
Q

How are Group I mGluRs involved in neuronal excitability? (effects on ion channels)

A

Postsynaptic Group I mGlu receptors mediate membrane depolarisation

Inhibition of K⁺ channels by Group I mGluRs leads to an increase in cell excitability (depolarising cell by blocking potassium channels responsible for leak conductance, or increase AP firing by interacting/inhibiting other potassium channels)

Increase activation of ionotropic NMDA receptors

58
Q

How are Group II + III mGluRs involved in neuronal excitability? (effects on ion channels)

A

Inhibition of Ca²⁺ channels (via Gio signalling) leads to inhibition of Glu release

Activating inwardly rectifying potassium channel (GIRK), helps to reduce NT release as hyper-polarising the neurones by opening the potassium channels

59
Q

What do we know about the membrane depolarisation that is mediated by group I mGluRs?

A

*NMDA, AMPA + ACPD (group I agonist) all cause rapid depolarisation

Prolonged depolarisation with ACPD (group I agonist) rapidly desensitised group I mGluRs on rat spinal motor neurons- but no effect on NMDA / AMPA depolarisations

If ACPD applied for longer period of time, then group I receptor activation tails off i.e. mGlu1/5 response is desensitised (wash off ACPD, comes back again)

60
Q

What mechanism may explain group I mGluR desensitisation?

A

Group I mGluR activated by ACPD (or DHPG): activates PLC-coupled receptor → G-protein mediated K+ channel inhibition (causes depolarisation)

  • PLC → DAG → PKC activation → leads to phosphorylation of receptors, feeds back and PHOSPHORYLATES Group I mGluRs → shifts into desensitised state
61
Q

How did Doherty et al (1997) show that group I mGluRs potentiate NMDARs?

A

Recorded potential difference across regions in hippocampus

Applied NMDA (short 1 min applications), then 10 mins CHPG (mGlu5 selective orthosteric antagonist).

CHPG did not depolarise alone but resulted in potentiation of NMDA-induced depolarisation in hippocampal CA1 neurons

Wash out CHPG - NMDA response returns to original size - TRANSIENT potentiation of NMDARs (not sure how it occurs)

*Drugs that potentiate mGlu5 may be useful to correct NMDAR hypofunction in schizophrenia

62
Q

What did Algarsamy et al (2005) show about Group I mGluRs and NMDARs?

A

Their interaction is BIDIRECTIONAL - adult rat slices, measured PI hydrolysis
with different concs of DHPG (Group I mGluR agonist).

  • NMDA won’t increase PI metabolism, but increasing concs of DHPG shows (some) increase, then starts to desensitise if over-activate receptors
  • NMDA + DHPG: potentiated the mGlu5 response (PI metabolism) - therefore mGlu5 activation leads to NMDAR potentiation and NMDA activation leads to mGlu5 potentiation
63
Q

How is NMDA-induced potentiation of mGlu5 responses (mGlu5 expressed in cell lines) thought to occur?

A

NMDAR causes DEPHOSPHORYLATION of the mGlu5 C-terminal tail

*remember: phosphorylating mGlu5 C-terminal tail causes mGlu5 desensitisation

64
Q

Describe the signalling pathways of Group II + III mGluRs

A

Classically couple to Gi/Go: inhibition of AC + decrease in cAMP + known to regulate ion channels via GβƔ subunits

GβƔ subunit activates K channels → hyperpolarisation, inhibits Ca channels → decreases transmitter release (classical signalling, there are also others)

mGlu7 can also activate PKC via interaction with both Gao and GβƔ → SELECTIVE blockade of P/Q-type Ca channels

65
Q

Why is the structure / signalling of mGlu7 different?

A

Intracellular C-terminal (Ct) tail has two important domains:

  1. Proximal binding site: binds GβƔ
  2. Extreme PDZ-binding motif: interacts with PKC interacting protein (PICK1)
    * similar: Group I interacting with Homer, mGlu7 can couple PICK
66
Q

What did Trepanier et al (2013) show about POSTSYNAPTIC mGlu2 / mGlu3 ?

A

NMDA current (30 min electrophysiology) in acutely isolated CA1 NEURONS

Control conditions: no change in current size. With ORTHOSTERIC mGlu2 AGONIST (LY…), NMDA RESPONSE POTENTIATED (enhances peak currents).

Enhancement occurs after agonist washout + persists for duration of recording (5 min agonist application = sustained NMDA potentiation)

Effect can be blocked by an mGlu2/3 antagonist

67
Q

What mechanism has been proposed to explain how mGlu2/3 can potentiate NMDAR currents?

A

Experiments with GluN2 selective antagonists suggest the NMDARs contain GluN2A:

Proposed model: postsynaptic mGlu2/3 inhibits cAMP-PKA pathway (Gi pathway) → activation of Src kinase (a tyrosine kinase) → phosphorylation of GluN2A NMDARs on CA1 neurons → potentiation of peak NMDAR current that persists after agonist washout

May be one of the mechanisms by which mGlu2/3 agonists act as antipsychotics (i.e. correction of NMDAR hypofunction)

68
Q

What is the mechanism by which presynaptic group II or III mGluRs act as autoreceptors?

A

Inhibit Glu release via G-protein mediated inhibition of Ca²⁺ channels

*GβƔ subunits of G-protein interact with ion channels, particularly pre-synaptic VGCCs (less Ca entry when AP reaches presynaptic terminal, less glutamate release)

This particularly occurs at mGlu7, but also at the other Group II/III receptors that are further away with sustained AP trains

Autoreceptor role serves to control the [Glu] at synapse + prevent excitotoxicity

69
Q

How does mGlu7a interact with Ca²⁺ channels?

A

GβƔ subunit interacts with VGCCs, but ALSO:

Activates Go + inhibits P/Q channel through PKC pathway (PKC activation inhibits P/Q channel, + decreases Glu release)

PICK1 PDZ domain interacts with mGlu7a extreme C-terminal domain. PICK1 also interacts with catalytic subunit of PKCα.

Dimerisation of PICK1 (via coil-coiled region) therefore links mGlu7a to PKC + keeps PKC in local vicinity - more likely that mGlu7a activation → PKC activation

70
Q

How has the role of PICK1 been demonstrated?

A
  1. KO PICK1: less regulation of Glu release with mGlu7 activation! Inhibition of P/Q Ca channels + synaptic transmission requires PICK1!
  2. PICK1 uncoupling causes absence-like seizures (see Bertaso et al (2008)

(Peptides conjugated to cell membrane transduction domain of HIV-1 Tat protein can crosses plasma membrane + BBB -

71
Q

How has the role of mGlu7 in epilepsy been demonstrated?

A

mGlu7 KO mice susceptible to epileptic agents

  • if decrease regulation of glutamate release by mGlu7, more likely to have some types of epilepsy in these animals
72
Q

What did Bertaso et al (2008) do?

A

Conjugated artificial peptides to cell membrane transduction domain of HIV-1 Tat protein so that they could cross plasma membrane and BBB

  1. Last 9 AAs of mGlu7 C-terminal (TAT-R7-LVI): inhibited interaction of mGlu7 C-terminal tail with PICK1 PDZ domain
    (because injected peptide preferentially binds PDZ of PICK1 instead of native mGlu7)
  2. Control peptide where LVI replaced by AAA (TAT-R7-AAA) not recognised by PDZ of PICK1 - no effect on native coupling
  3. Cortical EEG in rats + mice show concomitant epileptiform discharges from left + right cortex 30 mins after IV TAT-R7-LVI, but not TAT-R7-AAA - therefore absence seizures may be due to disruption of mGlu7/PICK1 coupling
73
Q

What are absence seizures?

A

Synchronised rhythmicity in thalamo-cortical circuitry resulting in non-convulsive seizures

Pharmacology of the nervous system (9 decks)

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