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Evidence for glutamate as a NT

1. Synaptic VGLUT transfers cytosolic glutamate into vesicle
(VGLUT = vesicular transporter on vesicles in pre-synaptic terminals- conc. Glu into vesicles)

2. NT released in Ca-dependent manner

3. Specific protein targets (ionotropic & metabotropic receptors)
- Postsynaptic: all 3 ionotropic & mGluRs
- Pre-synaptic: mGluRs, some evidence that KARs & NDMARs also expressed presynaptically (reasonably convincing but less strong)
- Glial cells (astrocytes): mGluR - surrounds synapses

4. Rapid removal of NT from synapse: glutamate transporters present on pre- and postsynaptic nerve terminals (EAAT = excitatory AA transporters
- (pre: EAAT2? unsure if exists, post: EAAT3)
- also on astrocytes (EAAT1/2)- variety of transporters expressed on glial cells, take glutamate into astrocytes

5. Process for glutamate synthesis: glutamate-glutamine shuttle and metabolic processes within presynaptic terminal

- enzyme pathway within pre-synaptic neuron where glutamine is converted into glutamate via glutaminase; then glutamate packaged into vesicles
- glutamate also cycled back from the astrocytes to the neuron so it can be used again
- glutamate not just a transmitter; also triggers cell death so can’t have high levels in brain (think stroke mechanism)
* to get glutamate from astrocyte → neuron, it’s converted to glutamine by the enzyme glutamine synthetase as this won’t activate cell death pathways
* glutamine then pumped out by glutamine transporter, shuttled across to neuron, taken up by glutamine transporter again, then converted back to glutamate (via glutaminase)


Main structure of AMPARs

Tetramers (4 subunits) assembled from GluA1, GluA2, GluA3 and Glu4:

Homomers (one type subunit) or heteromers (different subunits - any combination)

- GluA1-GluA2 relatively common type of heteromer

Differences in subunit composition in different brain areas (or neurons within brain region) leads to diversity in the functions and properties of AMPARs and in their sensitivity to pharmacological agents e.g. GluA3 highly expressed in some brain regions & less in others

Even in same regions, different types of neurons can express different combinations: drugs can be made to selectively target some subunits


AMPA and Kainate Receptor Subunit Topology?

(Topology = geometrical properties/spatial relations)

Consists of two dimers: e.g. 2 GluA1-GluA2 dimers come together to form tetramer

Amino terminal domain (ATD)
- extracellular
- contains clam-shell like domain (but nothing in particular binds it)

Ligand binding domain (LBD)
- Contains S1S2 domain, contains clam shell domain where GLUTAMATE binds
- S1 domain (extracellular peptide loop from TM1 → N terminal)
- S2 domain (extracellular peptide loop joining TM3 + TM4 & therefore the carboxyl terminals)

Transmembrane domain (TMD)
- contains pore of ion channel & intracellular carboxyl terminals
- note TM2 doesn’t go fully across the membrane; it forms a RE-ENTRANT loop which forms lining of PORE

TM2 has Q/R site very close to pore, determines calcium permeability

((All AMPA receptors: Na⁺ in & K⁺ out, some receptors will allow calcium in but not all))


What is RNA editing in AMPA receptors?

GluA2 DNA encodes for glutamine (before transcription)

Q/R switch: after mRNA produced, the triplet coding for glutamine (Q) is switched to arginine (R) in the mRNA

Edited gluA2 mRNA has arginine (R) at residue 607 (the Q/R site). This change is expressed at a site in RE-ENTRANT LOOP 2 in GluA2 that controls Ca²⁺ ion permeability through the ion channel.

RNA EDITING causes calcium IMPERMEABILITY: arginine doesn’t allow calcium through the pore

AMPA receptors containing EDITED GluA2(R), = Ca impermeable

AMPA receptors containing UNEDITED GluA2 (Q) OR LACKING GluA2 = Ca permeable

EDITED (GluA2(R)-containing receptors) also have lower single channel conductance
(how much charge channel will carries when activated). Therefore, editing means less current passes through


What kind of subunits do most AMPAs show? In what situations are different types found?

Most AMPARs in adult rodent brain = EDITED(R) GluA2, so Ca²⁺ IMPERMEABLE (most research = rodent but also seems true of adult humans). Ca²⁺ important second messenger- tightly controlled, therefore most AMPARs are Ca²⁺-impermeable

GluA2-LACKING AMPARs expressed:

- following induction of some forms of LTP
- chronic stress (↑proportion of AMPARs without GluA2, replaced with other subunits)
- early during development (first 2 postnatal weeks in rodents; synapses formed, more plasticity, AMPARs probably involved)
- some GABAergic interneurons

In disease e.g.
- after stroke, down-regulation of GluA2 - (unlikely to be helpful as calcium can trigger cell death?!)
- MND patients show decrease in GluA2 EDITING in motor neurons: may contribute to the death of the motor neurons (NOTE: this is less EDITING rather than less GluA2)


What are examples of AMPA antagonists?

CNQX: AMPA, Kainate (& NDMA) antagonist
*NBQX: AMPA selective
- COMPETITIVE: compete at LBD, not selective for specific subunits

Blake et al (1998): Electrophysiology: glutamate synapses between CA3 + CA1 in rat hippocampus slice (record post-synaptic cell, stimulate axons, record response).
- Control condition: depolarisation (glutamate-mediated. CNQX applied: block glutamatergic synaptic response. Wash drug away; response returns. (same happens with NBQX).


What kind of transmission at AMPARs?

Fast excitatory synaptic transmission mediated by AMPA receptors in most areas of brain (hippocampus and many other regions)

AMPA mediates majority of FAST BASAL synaptic transmission (everyday communication between brain regions)

(some regions: KainateRs also involved, AMPA key in hippocampus and other areas)


What is an antagonist at AMPARs WITHOUT EDITED GluA2?

*IEM-1460: antagonises AMPA receptors without edited GluA2

Magazanik et al (1997): recorded from GluA3 tetramers or GluA3/GluA2(R) tetramers (only receptors expressed on cell). Used kainate as agonist of AMPARs - causes inward current, partially reversed by 1um of IEM-1460.

Increase conc of IEM-1460 (2µm), more of a block - reverse when wash off antagonist

With GluA3, 1-2µm gives large ~50% block, but with GluA2(R) subunit, 100µm only causes about 10% inhibition


What is PTX?

Philanthotoxin (PhTX-74) from wasp venom
Antagonist in absence of GluA2

Enters pore & interact with Q/R site to block the ion channel, but won’t fit when GluA2 present

Poulson et al (2014):

Concentration-response curve, increasing concentrations of PhTx-74: for GluA1 homomer and GluA3 homomer, PhTx quite effective at reducing size of response (EC50 about 0.3mM)

If GluA2(R) present (with either GluA1 or GluA3), drug much less effective, about 100x change in potency (two log units shift to right)

*Therefore PhTX-74 can distinguish between AMPA receptors that contain GluA2 and those that don’t, useful in determining when we see these receptors


Main structure of KARs?

Tetramers (homomeric or heteromeric):
GluK1, GluK2, GluK3, GluK4 & GluK5

GluK1-3 = LOW affinity for kainate
GluK4-5 = HIGH affinity for kainate
(but less than 5x difference - not big diff)

GluK4 & GluK5 have an RXR RETENTION MOTIF in C-TERMINAL tail: when protein expresses motif, gets RETAINED in ENDOPLASMIC reticulum, never gets trafficked to cell surface

HOMOERMIC assemblies of GluK4 or GluK5 not surface expressed (also tetramers of GluK4+GluK5). However, GluK1-3 can combine with GluK4 or GluK5 to produce tetramers that are surface expressed (retention is overcome & receptor trafficked to surface)

*subunit composition varies in different brain areas, therefore differences in function/pharmacology in different brain regions


What is the function of KARs in CA1 of hippocampus?

CA1 (and other areas): KARs = presynaptic, DEPRESS glutamate transmission

Depolarise axon terminals, causing INITIAL SPIKE of glutamate release (AMPA EPSC amplitude increases)

BUT VG CALCIUM channels INACTIVATE with prolonged depolarisation → depolarisation block → less glutamate released → DEPRESSION in the AMPAR response (AMPA EPSC amplitude decreases)

*NS-102: kainate receptor antagonist, blocks this effect of kainate (AMPA EPSP amplitude is maintained despite kainate application)


What is the function of KARs in CA3 region of hippocampus?

CA3 = high density of POST SYNAPTIC KARs
Allow sodium influx (+maybe calcium) - depolarises cell contributing to EPSP

TRAINS of stimulation (rather than 1 stimulation causing 1 AP), e.g. 5 stimulations in short period → BIGGER GLUTAMATE RELEASE→ trigger a SLOW KAINATE RESPONSE (slow depolarisation lasting ~100ms (in AMPA, response over by 10-15ms)

This slow depolarisation is blocked by CNQX (AMPA/kainate antagonist)

LY294486: first selective(ish) kainate receptor antagonist - also suppresses this response
* EPSC = excitatory post-synaptic currents


How can Xray crystallography be used to visualise ionotropic receptors?

1) Grow part of the protein in solution; crystals start to form as protein precipitates & increases in size. Grow crystal of AMPAR.

2) Pass X-rays through crystal structure: results in diffraction of X-rays.

3) Measuring angles and intensities of the beams = = 3D picture of the electron densities (electron density map)

4) Mean positions of atoms can be determined from this, and their chemical bonds

- can be used to develop drugs that will selectively bind to different regions of the receptor (most common way is to take LBD with the agonist bound, grow a crystal of that and look at the structure)

Whole receptor much bigger protein, more difficult to do

Isolated ligand bind domain (LBD) structures of glutamate receptors are more common than those of the full tetramer - structures of membrane bound proteins difficult to obtain: correct protein folding outside the membrane difficult to achieve due to hydrophobic transmembrane domains


What is the crystal structure of antagonist bound GluA2 tetramer?

ATD has a clamshell structure and has a LOW SEQUENCE HOMOLOGY with BACTERIAL PERIPLASMIC proteins and mGluRs - it is involved in receptor assembly and trafficking.

LBD has a clamshell structure and is made up of S1 & S2 segments. The TMD has similar structural features to an inverted K⁺ channel pore.

- Structure of the C-terminal tail was not resolved (easier to get the crystals without the C-terminal, and not particularly long anyway, therefore don’t know structure of the tail)


What is the position of the LBD in ionotropic glutamate receptors? How do we know?

1) Bind agonist to LBD, then cut using RESTRICTION ENZYMES; cut particular regions e.g. top of transmembrane regions

2) Add a LINKER so that it stays as one protein, then CRYSTALLISE the region (very simplistic view because isolated bit of protein, but hopefully will relate to structure when it is in the complete protein)

- working out exact LBD structure/exactly where agonists bind - develop drugs that bind some receptors + not others?

- using molecular biological techniques, cDNA encoding for only the S1S2 REGION was isolated (gave access to large amounts of protein which could be crystallised, in some cases with agonists/antag. bound to S1S2 region


How can agonists and antagonists stabilise different LBD conformations?

If grow crystal with agonist bound, e.g. AMPA with GluA2, S1/S2 shut, CLAM SHELL CLOSES, causes ACTIVATION of receptor

If grow crystal with antagonist bound e.g. UBP282 with GluA2, clam shell domain is WEDGED OPEN, prevents activation/shutting of clam shell

*technique not dynamic - can’t see process of change between open/shut


What are the binding sites of AMPAR NAMs?

- NAMs antagonists but not bindings to LBD

- Bind close to ion channels at all 4 subunits; stop activation of receptor (binding of agonist to AMPAR/KARs causes closing of shell which causes TWISTING of receptor to open the ion channel: GYKI STABILISES the receptor to STOP ion channel opening.

GYKI & PERAMPANEL bind in TMD region adjacent to the PEPTIDE linker connecting the TMD to the LBD (bind to a site formed by pre-M1, M3 & M4 in each subunit - and to residue to an adjacent subunit - prevents movement of transmembrane segments necessary for pore opening
(i.e. 4 NAM binding sites per tetramer)

Prevents the movement of transmembrane segments necessary for ion channel pore opening.


What is perampanel?

Perampanel: AMPAR NAM

Anticonvulsant activity and is approved for treatment of patients with partial onset seizures (these affect only part of the brain at onset)


How have crystal structures aided design of potent GluK1 KAR antagonists? What has this provided insight into?

Insight into structural basis for KAR vs AMAPR selectivity

Willardine = AMPAR agonist

UBP310 = exactly the same, but with a thiophene ring = kainate selective agonist (very potent, 18nM = high affinity)

*For GluA2 + GluK2, affinity is over 100µM, therefore much more potent for some kainate receptors than others
* Most potent → least potent: GluK1 → GluK3 → GluK2 + GluA2 (AMPA)
*not good at all for GluK2/AMPA subunit


How is UB310 selective?

The THIOPHENE ring forms favourable interaction with a VALINE residue in GLUK1 to enhance binding affinity at the LBD

Valine residue in GluK1 replaced by leucine residue in AMPARs (e.g. GluA2)

-This larger residue in AMPARs impedes antagonist binding (leucine residue takes up some of the space where UB310 usually binds) - explains GluK1 vs AMPAR selectivity


How can the selectivity of UBP310 be improved?

When UBP310 binds LBD of the KAR, there is quite a lot of free space above it, because the S1S2 domain is hyperextended when UB310 is bound (Xray).

Produced a derivate of UBP310 → ACET; this has a phenyl (BENZENE) ring added to the thiophene to create a BIGGER structure - free space above UBP310 is occupied by the phenyl ring, makes it better at binding to GluK1 & GluK3, therefore the antagonist is becoming MORE selective for kainate receptors (fits kainate better to AMPA)


What is UBP161?

GluK1 selective antagonist

Fast application (100ms) of 30mM L-glut to HEK293 cells expressing GluK3 in a whole-cell electrophysiological assay is used to avoid densensitisation (see later lecture)

Producing currents in cells which are dependent on the diff subunits & looking at inhibition of those currents while increasing drug concentrations; UBP161 POTENT ANTAGONIST in GluK1 expressing cells, much weaker antagonist on GluK2 or GluK3


What techniques have been used to develop GluK1 selective antagonists?

Computer-aided drug design and point mutation studies: UBP161 docked in GluK1 LBD.

Hypothesised: HYDROGEN BOND interaction of a CARBOXYLIC ACID group (drug) with SERINE RESIDUE (protein), holds drug in LBD

In GluK3, serine residue is replaced by a HYDROPHOBIC ALANINE residue - hydrogen bond interaction is lost

*Is this why UBP161 is a weak antagonist on GluK3? (GluK1 = serine, GluK3 = alanine)


How can the selectivity of UBP161 be altered? Study to show this?

Cells with WT GluK3 & point mutated GluK3: alanine residue replaced by serine (A-S)

Competition binding assay: added tritiated kainate (H replaced with tritium); to preparation of membranes, then add UBP161 increasing concs.

UBP161 does NOT inhibit binding of [³H]kainate to WT GluK3 (increasing to 1mM does have affect, but this is a 10x increase!)

It DOES inhibit binding to the point mutated GluK3 (A-S)
Suggests that ALANINE residue in GluK3 is responsible for the LOW AFFINITY of UBP161

(Single AA has changed receptor from being low affinity antagonist to better anatg, still not as good as GluK1, but has helped to shift the binding)


What are the desensitisation properties of ionotropic glutamate receptors? (experiment)

Receptors expressed in HEK-293 cells: outside-out patches

*AMPARs + KARs show rapid desensitisation in response to sustained activation by (S)-glutamate - very rapid, inward as channel opened, then desensitisation although agonist still present


How do positive allosteric modulators work?

Bind different sites at receptors (not the LBD) & enhance their function by BLOCKING DESENSITISATION

Several drug classes block desensitisation of AMPARs

*CONCANAVALIN A blocks desensitisation of GluK1 or GluK2-containing but NOT homomeric GluK3-containing KARs

(carbohydrate binding protein isolated from plants)


What is flip/flop?

Different forms of AMPAR

GluA1-4 have region of ALTERNATIVE SPLICING that produces ‘flip’ and ‘flop’ forms of the subunits

Flip-flop region = short region in EXTRACELLULAR PEPTIDE DOMAIN JONING TM3 + TM4 (in the S2 domain, just before TMD)

Results in 9-11 amino acid difference for each subunit (reasonable size diff between flip and flop)

‘Flop’ versions of GluA2-4 DESENSITISE FASTER and RECOVER from desensitisation more SLOWLY

Flip/flop also affects sensitivity to POTENTIATORS, particularly cyclothiazide


What is a study of flip/flop in GluA2?

Koike et al 2000: GluA2 expressed in HEK cells

Flip: add glutamate: peak current, desensitisation, remove glutamate

Flop form: add glutamate, peak current, DESENSITISATION MUCH FASTER, remove glutamate, SLOWER RECOVERY

*remember: kainate = agonist at AMPA receptors, shows kainate as well as glutamate, kainate activates receptor but doesn’t cause the big peak, just provides low steady state of current (smaller response but doesn’t desensitise)

*note: affect of glutamate will be the same at Glu3 and Glu4


What is a study of flip/flop in GluA1?

Mosbacher et al (2004): GluA1 expressed in HEK293 cells

Flop form (o) + Flip form (i): no difference in desensitisation in response to glutamate

Flip-flop forms only affect desensitisation in GluA2-4


How does cyclothiazide affect AMPARs?

Partin et al (1995)

GluA1 (i) form: add glutamate, rapid desensitisation, CYCLOTHIAZIDE SLOWS DESENSITISATION

GluA1 (o) form, Cyclothiazide less effective
*Example from GluA1, but also true of GluA2, GluA3 and GluA4

Flip/flop difference in drug sensitivity not seen with other inhibitors of desensitisation (only cyclothiazide) *other drugs block desensitisation, but don’t show sensitivity between flip and flop

CYCLOTHIAZIDE binds at the flip/flop site (extracellular peptide chain joining TM3/TM4)


What are the QR site and the flip/flop site both examples of?

Post-transcription RNA editing


Flip/flop = extracellular peptide chain joining TM3 and TM4


What is Cryo-EM?

Cryo-electron microscopy

- modern technique for 3D structures of membrane bound proteins (which are not easily accessible using X-ray crystallography)

Beam of electrons fired at frozen protein solution. Emerging scattered electrons pass through lens to create magnified image on detector. Detector works out structure (electron beam --> frozen sample --> lens --> electron detector)

*Studies underway to enhance resolution by improving electron detectors and methods of preparing protein samples; best resolutions to date = 2.2-3.4 angstroms


What is different between Cryo-EM and X-ray crystallography?

Take protein sample, isolate domain / membrane, apply drug, then freeze sample: happens QUICK, fixed receptor in particular state

To see diff possible combinations when drug is bound; need more real time rather than days/weeks of precipitation (X-ray not dynamic - bind drug then prp.)

Cryo-EM can capture ensemble of protein conformations including resting, open and desensitised states


How have techniques revealed mechanism of ionotropic glutamate activation + desensitisation?

Combining cryo-EM & higher resolution X-ray crystallog. of GluA2 + GluK2 tetramers has led to model of channel opening & desensitisation


Describe the model for channel opening in ionotropic glutamate receptors?

Glu binding leads to LBD closure and CORKSCREW ROTATION that opens channel

Rotation causes the UPPER LOBES of LBDs to PULL DOWN the ATDs

The LOWER LOBES of the LBD exert LATERAL and UPWARD forces to OPEN the channel


Describe the model for desensitisation in ionotropic glutamate receptors?

STRAIN in open state RELIEVED by transition to desensitised state

Desensitised state accommodates closed LBDs (which lead to increased tension) a closed channel (which requires release of tension)

This is achieved by changing the SYMMETRY of the arrangement of the LBD in the tetramer to MATCH that of the ion channel in its closed state

(Agonist binds: clam shell shuts: causes region of protein to twist + pulls the channel open; this puts strain on the TMD, protein wants to return to a more relaxed state (lower tension. LBD twists around even further, puts channel into desensitised state- actually thought to be several different desensitised states)


What did Sun et al (2002) show about cyclothiazide?

GluA2 subunits expressed on HEK cells: add CTZ: no desensitisation (completely blocked desensitisation)

X-ray crys. of GluA2(i) showed that CTZ blocks desensitisation by binding to dimer interface.

2 CTZ molecules bind in equivalent positions in LBD dimer interface between two adjacent GluA2 subunits. *2 molecules bound for each dimer

Each CTZ molecule spans the interface thereby STABILISING it and PREVENTING CONFORMATIONAL CHANGE that leads to desensitisation
*allows initial closure of LBD shut, therefore allows protein to do initial twist, but then stabilises structure with channel open + doesn’t allow it to move → prevention of desensitisation (stops channel closure)


What did Jim et al show?

GluA2 (flop) expressed in HEK293 cells

Rapid (sub ms) agonist pulse won't desensitise receptor: causes DEACTIVATION (agonist diffuses away from receptor and channel shuts, goes back into resting state)

Continuous (500ms) glutamate application causes opening of channel then desensitisation


What is CX614?

PAM - slows desensitisation more profoundly at GluA2(o) than CTZ

Jin et al (2005): GluA2 flop in HEK293 cells

CX614: SLOWS DEACTIVATION but ALSO slows desensitisation

CTZ: slows DESENSITISATION but no affect on deactivation

2 types of potentiator; CX614 slows desensitisation and deactivation, CTZ only slows desensitisation


Where do modulators that slow deactivation bind?
(e.g. CX614)

PROLINE (top of hinge) & SERINE residues (bottom of hinge): HINGE region of DIMER INTERFACE between 2 GluA2 LBDs

Hinge = pivot point for opening and closing of the LBD

*Stabilises conformation where LBD shut - slows deactivation by keeping agonist in closed clamshell

Binding regions for CX614 + CTZ overlap, therefore CX614 may also be involved stabilising open channel state (so it slows desensitisation like CTZ)


Summary of PAM actions:

Stabilisation of dimer INTERFACE primarily affects receptor DESENSITISATION

Stabilisation of agonist bound CLOSED-CLEFT state of LBD by binding the HINGE region at the dimer interface slows deactivation

CX614: primarily affects deactivation, but can also block desensitisation

Overlapping activities on desensitisation and deactivation are due to an overlap of the sites to which positive allosteric modulators bind to affect these properties

Unknown whether drugs selective for deactivation/desensitisation or drugs affecting both would be most useful therapeutically


Why interested in PAMs rather than just adding agonist?

Don’t want receptors activated all the time as could lead to cell death, this method ensures it affects the specific subunit; enhancing natural patterns of activation


What are therapeutic potentials of AMPA PAMs?

Ampakines = positive AMPAR modulators (no cognitive enhancers on market yet - company (CX614) now focusing on respiratory depression / sleep disorders)

Nootropics: facilitate learning & memory (smart drugs)

Clinical studies: treating COG DEFICITs in Parkinson’s, AD & schizop. (promising results)

SLEEP-DEPRIVED healthy volunteers: improved performance in memory, alertness, reaction time & problem solving ability

Clinical trials: Prevention of opiate-induced respiratory depression (without affecting analgesic activity)

SLEEP APNOEA: think there are phase III trials stimulation of breathing...?? *reading

RAPID ONSET ANTIDEPRESSANTS: Increase levels of BDNF, (promising in animal models of depression)


What are auxillary proteins?

Transmembrane proteins that modify properties of AMPARs + KARs

Neto1 & Neto2 interact with KARs

TARPs interacts with AMPARs


How has the role of Netos been studied?

KO Neto1:
- Mossy fibre → CA3 synapse: KAR synaptic component loses its slow kinetic profile & KARs desensitised more quickly (so KAR EPSC looks more similar to the fast AMPA EPSC)

If can change desensitisation/deactivation - potential drug target?


What are TARPs?

Transmembrane AMPAR regulatory proteins - family of 8

Ɣ-2, Ɣ-3, Ɣ-4 & Ɣ-8 most closely related and interact with AMPA receptors

Ɣ-2 = stargazin (discovered first)
*KO produces stargazer phenotype; similar to absence epilepsy in humans (dyskinesia, ataxia, head-tossing)

*KO also has no AMPARs in cerebellar granule cells (Ɣ-2 needed for functional expression of AMPA receptors in cerebellar granule cells - interact with AMPARs - function + getting to cell surface)

((Ɣ-1 = interacts with 1 of the VGCCs (lots of proteins interact with subunits of VG calcium channels)))


What did Jackson & Nicholls (2011) show?

TARPs help traffic AMPARs to surface

HEK cells can express GluA1 without TARPs: TARPs SLOW DEACTIVATION & DESENSITISATION (affects kinetics & binding affinity)

CNQX is HIGHLY effective without TARP, but not with TARP

PhTx (channel blocker) - effective when TARP PRESENT, but not when TARP absent!


What did Watanabe (2013) show?

Subunit expression of TARPs:

*First one to be induced = gamma 2, highly expressed in the cerebellum
*Gamma 8, interacts with AMPA, highly expressed in hippocampus and cortex, not in cerebellum/brainstem
*Can use this to develop selective drugs that target AMPA receptors in different places


How does the glutamate transporter work? Significance?

1 Glu in = 3 Na⁺ & 1H⁺ in, 1K⁺ pumped out, therefore, electrogenic transporter (charge change as Glu taken in)

Rate of Glu transport will be dependent on some ions (could be affected when something goes wrong in the brain e.g. stroke)

If record electric currents from glial cells: shows glutamate transport into the glial cell because change in membrane potential when the transporter is functioning; used as a measure Glu release from pre-synaptic terminal

*more glutamate released → greater charge difference when glutamate is taken up into the glial cell


Evidence for AMPAkines as nootropics??

Facilitate synaptic transmission in hippocampus (and other brain regions): improves performance in ANIMAL MODELS of LEARNING & MEMORY
- Subset (NOOTROPIC agents): facilitate learning and memory: ‘smart drugs’


Evidence for AMPAkines in ADHD?

Phase II clinical trial of CX717 (ampakine) demonstrated preliminary efficacy in ADHD, but halted due to neurotoxicity concerns. CX717 also shown to halt opioid respiratory depression in human volunteers.

April 2018 - paper released to show that the vacoules forming in white matter were only formed post mortem(artefact of tissue processing) - not a problem in vivo