amino acid transporters elm1

how is glutamine converted to glutamate

• glutaminase

how is glutamate converted to glutamine

glutamine synthetase

how is glutamine converted to GABA

• glutamine → glutamate (glutaminase)

• glutamate → GABA (GAD)

role of astrocyte in glutamatergic synapses

• glutamate is removed from extracellular space, taken up by EAAT

• converted to glutamine by glutamine synthease

• safely exported via glutamine transporters

• presynaptic nerve terminal also expresses glutamine transporters

• recycled for glutamate synthesis

glutamatergic synapse general steps

1. glutamate synthesis - glutaminase

2. packaged into vesicles by vesicular glutamate transporter VGLUT

3. glutamate activates postsynaptic receptors

4. termination of glutamatergic signaling through excitatory amino acid transporters EAAT

membrane topology of pLGIC

4 transmembrane domains

2nd domain lining the ion conducting pore

membrane topology of iGluR

• contain 3 transmembrane domains

• pore forming region is created by membrane dipping, re entrant, loop located between the 1st and 2nd transmembrane domain

agonist binding site of pLGIC

• formed by residues contributed by the N terminal extracellular domain of neighbouring subunits

agonist binding site of iGluR

• found within venus fly trap

• 2 lobes close around agonist

• one lobe is formed by residues within N terminal

• one lobe is formed by extracellular loop between 2nd and 3rd transmembrane domain

NMDA subunit composition

• assembled from subunits designated GluN1, GluN2A to GluN2D, GluN3A and GluN3B

• contain 2 copies of GluN1 which provide glycine binding sites

• GluN3 subunits can be incorporated - form tri heterometric receptors

AMPA receptor subunit confirmation

GluA1-4

Kainate receptor subunit composition

• GluK1-5

• GluK1-3 form homomeric and heteromeric receptors

• Gluk4-5 cannot form functional homomeric receptors and instead only heteromers in combination with GluK1-3

ampa distribution and function

• most common iGluR found in the cns and widely distributed in the brain

• permeable to sodium, potassium and some calcium

• mediate fast synaptic transmission

• 4 glutamate binding sites but only 2 to be bound for the channel to open

which ampa receptor subunits are permeable to calcium

• GluA1, GluA3 and GluA4 - highly permeable to calcium

• rare in adult brian

most common subunit confirmation in the cns for ampa receptors

• GluA2 which forms a heterotetramer with GluA1 or GluA3

explanation to ampa receptors being impermeable to calcium

• GluA2 subunit

• contains arginine residue at 607 instead of glutamine

which GluA subunit has roles in autism, epilepsy and intellectual disability

GluA2

NMDA receptor subunits

2 x GluN1 subunits which bind glycine

2 GluN2/N3 which bind to glutamate

binding sites of NMDA receptor and targeted by what

• the glutamate-binding site, which can be targeted by agonists and antagonists

• the glycine-binding site, which can also be targeted by agonists and antagonists

• a polyamine-binding site, which mediates both positive and negative allosteric modulation

• a channel-blocker binding site within the pore of the receptor

conditions where iGluR are implicated

• Schizophrenia

• Alzheimer's disease

• Parkinson's disease

• Autism spectrum disorders

• Stroke

• Neuropathic pain

• Depression

• Epilepsy

group i mglur

• compromise mGluR1 and mGlur5

• predominately postsynaptic

• located on neuronal cell bodies and dendrites

• coupled to Gq proteins

• enhance NMDA receptor function and can inhibit potassium currents

• potentiate inhibitory currents mediated by GABA-A receptors

group ii mGluR

• mGluR2 and mGluR3

• coupled to Go

• found at presynaptic site

• inhibitory autoreceptors or heteroreceptors, reducing neurotransmitter release

group iii mGluR

• mGluR4, mGluR6, mGluR7, mGluR8

• coupled to Gi, presynaptic, inhibitory like group II

Conditions in which mGluR-based therapies have been explored, or may hold therapeutic potential, include:

• anxiety disorders

• depression

• schizophrenia

• substance use disorders

• chronic pain

• Parkinson’s disease

potential cognitive enhancers for mglur

• positive allosteric modulation of group 1 and group 2 receptors

• pos effects on synaptic plasticity and learning, still in pre clinical studies

what is a histological marekr for GABAergic neruons

• GAD

• expression is restricted to inhibitory neurons

GABAergic synapse

1. GABA is packaged into synaptic vesicles by vesicular GABA transporter

2. GABA can activate postsynaptic receptors

3. removed from synaptic cleft by high affinity GABA transporters (GATS)

4. once inside the astrocyte, GABA enters citric acid cycle, converted to glutamate, glutamine (glutamine synthetase), safely exported via glutamine transporters

5. presynaptic terminal expresses complementary glutamine tranpsorters GlnT, allowing glutamine to be taken up and reused

where are GABA transporters (GATS) expressed

both presynaptic terminal and neighbouring astrocytes

which receptor stabilizes the E-Cl?

GABA-A receptor

use of drugs to enhance GABA-A?

• sedatives

• anxiolytics

• anticonvulsants

• anaesthetics

how does the chloride gradient differ from adults to neonatal and foetal brain tissue?

• in adults the ECl is close to rmp

• in neonatal the ECl is more positive than the rmp

effect of GABA-A receptor activation in neonatal brains?

• leads to chloride efflux

• membrane depolarisation rather than hyperpolarisation

How many distinct drug binding sites for GABA-A receptors?

• at least 5

subunits of GABA-A

• 19 different subunits

• but expression is limited to a few dozen confirmations

what is the most common GABA-A confirmation

• 2 alpha

• 2 beta

• single gamma subunit

Predominant synaptic GABA-A receptor subtype in the brain?

• two alpha 1

• two beta 2

• one gamma 2

Where is the binding site for GABA

interface between beta subunit and neighbouring alpha subunit

Benzodiazepines

CNS depressant drugs for short term severe anxiety, insomnia or acute seizures

benzodiazepine high affinity binding site

• between gamma 2 and adjacent alpha subunit of GABA-A

Extrasynaptic GABA-A receptors

• located outside synapse

• maintain steady, tonic level of inhibition in the cns

• respond to low, persistent ambient levels of GABA present in the extracellular space

• most contain delta subunit instead of gamma

• sensitive to general anaesthetic agents

Why are benzodiazepines not effective for all GABA-A receptors?

• benzodiazepine has high affinity binding site next to gamma 2 and alpha subunit

• most extrasynaptic receptors contain delta subunit instead of gamma 2 so are insensitive

location and effect of GABA-B receptors

• presynaptically - inhibit neurotransmitter release, autoreceptors or heteroreceptors

• postsynaptically - opening of potassium channels, leading to hyperpolarisation

subunits of GABA-B

• subunits associate via c terminal intracellular tails

• GABA-B1 subunit contains GABA binding site

• GABA-B2 is responsible for coupling the receptor to the Gi proteins and initiating intracellular signalling

How are GABA-B receptors pharmacologically distinct from GABA-A?

• insensitive to allosteric modulators such as benzodiazepines and barbiturates

• activated by muscle relaxant baclofen instead

What does baclofen treat?

• cerebral palsy

• multiple sclerosis

What regulates the uptake of glycine?

• glycine transporter

• found in presynaptic terminals and surrounding astrocytes

glycine receptor structure

• pentameric ligand gated ion channel

• alpha 1-4 and beta

• 4 transmembrane domains

• 2nd transmembrane domain lining the ion conducting pore

function of alpha subunit of glycine receptor

• form functional homomeric receptors

agonist binding site of glycine receptor

• interface between alpha and beta subunit

alpha 1 glycine receptor

• found in the spinal cord and brainstem

  • mediate fast inhibitory transmission, but are also present in some brain regions

alpha 2 and 3 glycine receptors

• expressed mainly in the brain, more restricted than alpha 1

alpha 4 subunit of glycine receptor

• non functional

• does not contribute to glycinergic signalling

renshaw cells

• inhibitory glycinergic interneurons located in the spinal cord

• regulate firing of motor neurons, strength and timing of skeletal muscle contraction

activation and inhibition of renshaw cells

• activated by collateral branches of alpha motor neuron axons

• feedback inhibition via strychine sensitive glycine receptors, limits motor neuron firing

• form inhibitory synapses with 1a inhibitory interneurons

1a inhibitory interneurons

suppress activity of motor neurons innervating the antagonistic muscle

Strychnine

• competitive antagonist at glycine receptors

• blocks the inhibitory actions of glycine released from renshaw cells and 1a inhibitory interneurons in the spinal cord

  • unchecked motor neuron activity, leading to severe muscle spasms and rigidity

oposthotonus

• body becomes rigidly arched, ultimately causes death by asphyxia as it disrupts the normal respiratory muscles too

low affinity agonists at glycine receptora

• taurine and beta alanine

GPR158

• GPCR like receptor

• indirectly regulates activity of g protein alpha subunits through RGS7/GB5 protein

Lytico-bodig - guam disease

• toxin - beta-methylamino-L-alanine BMAA within the seeds

  • when cooked there are trace amounts of the toxin, but seen in bats

• agonist to all three types of ionotropic receptor NMDA, kainate and AMPA

• excitotoxicity

Lathyrism

• disease of famine

• found in lathryus plans - glutmate analogue called oxalyldiaminoproprionic acid OADP

• potent agonist of AMPA type ionotropic glutamate receptors

• most severe in lower part of body and can result in paralysis

what can mGlu receptors form heterodimers with?

• non mGlu receptors such as 5-HT2A receptor

  • connected by disulfide bridge across the extracellular domain of each protein

what is GABA transport inhibited by?

• tigabine

• used to treat epilepsy

What inhibits GABA → glutamate?

• vigabatrin

• used in epilepsy treatment

Where do drugs bind to GABA-A receptors?

• the GABA binding site

• allosteric modulatory sites

• the ion channel pore