Glutamate Flashcards
Where is glutamate found?
It is a universal cellular constituent meaning that it is found in all cells.
Is glutamate an essential amino acid?
No it is not an essential amino acid-derived from the diet as it can be synthesised by neurons within the pre-synaptic neuronal terminal.
How is glutamate synthesised?
As either a by-product of the tricarboxylic acid cycle/Kreb cycle from alpha-ketoglutarate or alpha-oxoglutarate by the enzyme GABA-transaminase.
Or it can be synthesised from glutamine from glutaminase.
Therefore the two key enzymes involved in glutamate synthesis is GABA-transaminase and glutaminase.
What are the two main functions of Glutamate?
Participation in the metabolic cycle (Kreb cycle) or acting as a neurotransmitter. Neurons compartmentalise these two types of glutamate so they can be used for different functions.
Describe the process of the synthesis, storage and release of glutamate.
Within the pre-synaptic terminal glutamate is synthesised by the enzyme glutaminase which converts glutamine to glutamate. Glutamate is uptake into storage vesicles by the action of vesicular glutamate transporter. The neurotransmitter remains in these storage vesicles until they are released by fusion with the synaptic membrane by an influx of calcium into the synaptic terminal in which the neurotransmitter then undergoes calcium mediated exocytosis. Once released into the synaptic cleft glutamate is then able to act on receptors on the post-synaptic membrane before its signalling is terminated by being uptaken back into the pre-synaptic terminal or astrocyte by the the action of the excitatory amino acid transporter which is sodium dependent. If uptaken into the astrocyte glutamate is converted to glutamine by the action of the enzyme glutamine synthase and is then released by glutamine transporters and uptaken by glutamine transporters expressed on the neuron. Glutaminase can then convert the glutamine back into glutamate.
What are the different types of glutamate receptors?
There are two main types of glutamate receptors:
Metabotropic glutamate receptors (which are GPCRs)
Ionotropic glutamate receptors which are ligand gated ion channels, which can be further subdivided into:
NMDA
AMPA
Kainate receptors
What are the similarities and differences between the different types of ionotropic glutamate receptors?
NMDA, AMPA and Kainate receptors all respond to the endogenous ligand, being L-glutamate. However they are distinguished all they all respond differently to synthetic analogues (e.g. NMDA receptors only respond to NMDA etc.).
ALL three have a similar receptor structure with four subunits coming together in a tetrameric organisation.
The receptors have different pharmacological, biophysical properties and can be expressed in different neurons and are responsible for different types of electrical response.
Describe the different subunits that can compose an NMDA receptor.
Although each ionotropic glutamate receptor is composed of only four subunits forming a tetramer, NMDA receptors are assembled for seven different potential subunits encoded by seven different genes. However it is formed as a hetero-tetramer formed of different subunits.
The potential subunits are GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A, GluN3B.
Typically 2 GluN1 and 2 GluN2 subunits come together forming a tetrameric ion channel complex.
How many different variants of the GluN1 can be formed?
Despite only being encoded by one gene, alternative splicing which has resulted in the identification of eight different subunits of GluN1 being formed.
Describe an NMDA subunit structure.
Each subunit within the tetramer of the NMDA ionotropic receptor consists of a large extracellular domain which includes the N-terminal domain (amino terminal domain) and the ligand binding domain where the endogenous agonist glutamate binds. In addition to three full transmembrane spanning alpha helical domains and one and re-entrant P-loop called M2 and an intracellular C-terminus (CTD).
Do the subunits used to compose the NMDA receptor affect its properties?
Yes which GluN2 subunits used to compose the NMDA ligand gated ion channel affects the certain properties including:
How the receptor is activated by the ligand
How long the receptor remains open once activated
How the ion channel is regulated by ions
How ions move through the receptor
Pharmacological differences
Compare the different properties of a NMDA receptor containing 2 GluN2A subunits to that containing 2 GluN2D subunits?
GluN2A has a much lower agonist potency meaning that are greater concentration of glutamate is required to activate the ionotropic receptor in comparison to GluN2D.
GluN2A also has a much faster deactivation rate so the channel closes much quicker compared to GluN2D. However the rate of conductance of GluN2A is much faster than GluN2D meaning that a greater concentration of ions are able to pass through the receptor in the same amount of time.
GluN2A also has a much higher calcium permeability and magnesium sensitivity.
What do patch clamp recordings show?
Patch clamp technique involves the study of ion flow in excitable cells such as neurons. It involves using a micropipette and creating a suction across one ion channel. An electrical current is applied and as the ion channel opens, ions flow into the micropipette generating a current which is then shown on the graph as a downward spike.
Describe the different ion channel responses when glutamate is applied to AMPA, Kainate and NMDA receptors.
When glutamate is applied to both AMPA and Kainate receptors there is rapid influx of ions into the receptor creating an inward current (shown as a downward spike on the graph) however both receptors are quickly deactivated, with the kainate receptor slightly slower than that of the AMPA.
However with NMDA the rate of desensitisation and deactivation is dependent upon the subunits present, but overall are slower compared to the others.
Describe the differences in ion channel responses between NMDA receptor subtypes.
Ionotropic NMDA receptors composed of either GluN2A or 2B subunits are quite quickly deactivated even when glutamate is continued to be applied whereas if containing GluN2C or 2D receptors expressing these have a sustained response to glutamate and do not deactivate quickly.
Which type of ions are able to flow through NMDA receptors?
Cation selective so monovalent cations such as sodium, potassium are able to flow through in addition to calcium, NMDA receptors in particular are noted for their high receptor permeability for calcium.
What are the five NMDA receptor binding sites?
- Glutamate (agonist site)
- Glycine site (modulatory site)
- Polyamine binding site (modulatory site)
- Mg2+ site (found within in the ion channel pore)
- Channel blocking site (found deep within in the ion channel pore residing within the plasma membrane)
Describe how NMDA receptors can be activated.
NMDA receptors are activated by two molecules of glutamate binding to the agonist binding site. NMDA the synthetic agonist can also bind to this site causing the activation of the receptor.
The EC50 for the receptor is between 0.5 to 3.3 micromol of glutamate and as seen previously this is dictated by the GluN2 subtype which determines agonist potency.
However co-agonists are required for full activation.
What are the co-agonists at the NMDA receptor?
Glycine which binds at the co-agonist binding site. Again two molecules of glycine are required for full activation.
The EC50 of glycine is 1 micromol.
However D-Serine and D-alanine can also act as co-agonists.
Name some competitive antagonists that act at either of the agonist binding sites on the NMDA receptor.
At the glutamate binding site D-AP5 there is limited drug development due to conservation of glutamate binding site between NMDAR, AMPAR, KainateR
At the glycine binding site kynurenic acid and CGP 61594, this reduces the extent of activation of the receptor.
Describe the NMDA receptor voltage dependent channel block.
At resting membrane potential, the ionotropic NMDA receptor is blocked by the binding of magnesium to its receptor binding site which is located deep within the ion channel pore. This prevents other ions entering the ion channel and so the channel is classified as closed. However when the resting membrane becomes more positive (is depolarised) to -30mV magnesium then exits its ion channel binding site which then enables the influx of other ions into the receptor pore.
However this relies on extracellular magnesium being present, without extracellular magnesium the NMDA receptor will be active even at negative membrane potentials.
Where is the polyamine binding site located?
Polyamines bind to modulatory site which is located in the ATD (amino terminal domain).
What are some examples of polyamines?
Spermine, spermidine and putrescine
What are the effects of polyamines binding?
Polyamines upon binding to their modulatory site has a positive allosteric effect as it induces a conformational changes which enhancing the effect of the agonist or co-agonist binding which results in enhanced affinity and enhanced ion channel responses to either the glycine or glutamate.
What is the effect of Ifenprodil?
Binds close to the polyamine site and has a negative allosteric effect making it more difficult upon glutamate or glycine binding for a conformation change to be induced which then enables the opening of the ion channel pore.
What do channel blocking drugs rely on for activity?
These drugs bind to a site deep within the ion channel pore which requires the ion channel to already be open/activated. Therefore they are also known as open channel blocking drugs or use dependent blockers.
