• GluA2

    Concentration dependent activation of GluA2 receptors by L-Glutamate, measured on the SyncroPatch 384PE

GluA2 | GluR2 | Glutamate Receptor Ionotropic, AMPA 2

Family:
Glutamate Receptors

Subgroups:
The Glutamate receptor family has further been divided into ionotropic and metabotropic glutamate receptors. 
The metabotropic glutamate receptor subfamily contains 8 members (mGluR1 - mGluR8)
The ionotropic glutamate receptors subfamily contains 16 members: 
AMPA receptors: GluA1 (GluR1) - GluA4 (GluR4)
Kainate receptors: GluK1 (GluR5), GluK2 (GluR6), GluK3 (GluR7) , GluK4 (KA-1), GluK5 (KA-2)
NMDA receptors: GluN1 (NR1), GluN2A (NR2A), GluN2B (NR2B), GluN2C (NR2C), GluN2D (NR2D), GluN3A (NR3A), GluN3B (NR3B)

Regulation and Function:
Ionotropic glutamate receptors are ligand-gated nonselective cation channels that allow the flow of K+, Na+ and sometimes Ca2+ in response to glutamate binding. Metabotropic glutamate receptors belong to the subfamily C of G protein-coupled receptors. Glutamate receptors are responsible for the glutamate-mediated postsynaptic excitation of neural cells, and are important for neural communication, memory formation, learning, and regulation.

GluA2: Background Information

GluA2 is a receptor for glutamate that functions as ligand-gated ion channel in the central nervous system and plays a a major role in excitatory synaptic transmission. It is activated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), L-glutamate and kainate in the order AMPA (quisqualate) > glutamate > kainate. The GluA2 protein is encoded by the GRIA2 gene and four subunits are required to form a functional channel. Each subunit has 4 distinct domains: an extracellular amino acid terminal domain (ATD); the extracellular ligand binding domain (LBD); the transmembrane domain (TMD) with 3 transmembrane segments (M1, M3 and M4) and 1 cytoplasmic facing re-entrant loop (M2); and an intracellular carboxyterminal domain. Binding of the excitatory neurotransmitter L-glutamate induces a conformation change, leading to the opening of the cation channel, and thereby converts the chemical signal to an electrical impulse. The receptor then desensitizes rapidly and enters a transient inactive state, characterized by the presence of bound agonist. In the presence of CACNG4, CACNG7 or CACNG8, the receptor shows resensitization which is characterized by a delayed accumulation of current flux upon continued application of glutamate. The subunit encoded by the GRIA2 gene is subject to RNA editing (CAG->CGG; Q->R) within the second transmembrane domain, which is thought to render the channel impermeable to Ca2+. Human and animal studies suggest that pre-mRNA editing is essential for brain function, and defective GRIA2 RNA editing at the Q/R site may be relevant to amyotrophic lateral sclerosis (ALS) etiology. Alternative splicing, resulting in transcript variants encoding different isoforms, (including the flip and flop isoforms that vary in their signal transduction properties), has been noted for this gene. AMPA receptors mediate fast excitatory synaptic transmission and play a role in hippocampal synaptic long-term potentiation and depression.

Gene:
GRIA2

Human Protein:
UniProt P42262

Tissue:
Brain, Pancreas: Bipotent Endocrine/Duct Progenitor cells, Spinal cord: Oligodendrocyte-like cells, Skeletal muskle: Limb Muscle Progenitor cells

Function/ Application:
Chemical synaptic transmission, ionotropic glutamate receptor signalling pathway

Pathology:
Status Epilepticus, Lateral Sclerosis, motor neuron disease, schizophrenia, amyotrophic lateral sclerosis 1

Accessory subunits:
CACNG4, CACNG7, CACNG8, GRIP2, CSPG4, TARPs

Interaction:
MMP4, ATAD1, PICK1/PRKCABP, GRIA1, SYNDIG1, LRFN1, CACNG5, SNX27, OLFM2, AP4B1, AP4E1, AP4M1

Forms complex with:
GRIA1, GRIA3, GRIA4, CNIH2, CNIH3, CACNG2, CACNG3, CACNG4, CACNG5, CACNG7, CACNG8; NSG1, GRIP1, STX12

Agonists:
Glutamate, AMPA, (S)-5-fluorowillardiine, ATPO, GYKI53655, GYKI53784, tezampanel, NBQX

Modulator:
Argiotoxin, LY404187, LY392098, cyclothiazide, aniracetam, CX516, CX546, IDRA-21, LY503430, piracetam, S18986

Assays:
Patch Clamp: whole cell, ultrafast extracellular perfusion

Recommended Reviews:
A nomenclature for ligand-gated ion channels. Neuropharmacology 56:2-5, Collingridge G.L. et al. 2009

Disease Areas

AMPA receptors have been implicated in depression, Parkinson's disease, Huntington's disease, ischemic stroke and neurodegenerative diseases such as dementia and Alzheimer's disease.

Data and Applications

AMPA Receptor (GluA2) - Activation by Glutamate

icon sp96   GluR2 PE DataSyncroPatch 384PE data and applications:
Cells were kindly provided by SB Drug Discovery.

The AMPA receptor (GluA2) was activated using different concentrations of glutamate (1 µM - 100 µM). Measured on the SyncroPatch 384PE the whole cell patch methodology and multi-hole chips were used.
The lower two images are displaying screenshots of single cell currents after repetitive glutamate applications:
Left: The same concentration of Glutamate was applied three times.
Right: Four different Glutamate concentrations were applied in a cumulative manner.

AMPA Receptor (GluA2) - Pharmacology

icon sp96   GluR2 PE Data PharmacologySyncroPatch 384PE data and applications:
Cells were kindly provided by SB Drug Discovery.

The AMPA receptor (GluA2) was analyzed using different positive and negative allosteric modulators (CNQX, LY404187, LY395153, CP465022, Cyclothiazide). After activating the receptor by application of Glutamate, the modulating compound plus glutamate was applied afterwards. Measured on the SyncroPatch 384PE the whole cell patch methodology and multi-hole chips were used.
The lower images on the left hand side are displaying a screenshot of a current after application of the positive modulator LY404187. The EC50 was determined as 379 nM.

AMPA Receptor (GluA2) - Cumulative Concentration Response

icon sp96   GluA2 GluCRC SP384PE SBSyncroPatch 384PE data and applications:
Cells were kindly provided by SB Drug Discovery.

The AMPA receptor (GluA2)was activated by increasing concentrations of glutamate on the SyncroPatch 384PE. L-glutamate was applied for approximately 500 ms in increasing concentrations (A) and a cumulative concentration response curve for glutamate was constructed for 222 wells (C).
The online analysis values peak amplitude and area under the curve (AUC) are shown versus time in Panel B. The fast activation of GluA2 could be captured at higher concentrations (inset; 1 mM).

AMPA Receptor (GluA2) - Inhibition by CNQX

icon pl  GluA2_CNQX_SB_PLPatchliner data and applications:
Cells were kindly provided by SB Drug Discovery.

The AMPA receptor (GluA2) was blocked by CNQX on the Patchliner. CNQX was pre-incubated and then co-applied with glutamate. CNQX blocked the GluA2-mediated response in a concentration dependent manner and the potency was dependent on glutamate concentration (left). Exemplar GluA2-mediated responses are shown on the right activated by 100 µM glutamate and inhibited by increasing concentrations of CNQX.

AMPA Receptor (GluA2) - Fast Perfusion with the Port-a-Patch

icon pap   Port-a-Patch data and applications:
Cells were kindly provided by SB Drug Discovery.

GluA2 GluCRC PaP SB

The AMPA receptor (GluA2) was activated by increasing concentrations of glutamate on the Port-a-Patch. L-glutamate was applied for approximately 500 ms in increasing concentrations (left) and a cumulative concentration response curve for glutamate was constructed for 8 cells (right). The fast activation of GluA2 could be captured at higher concentrations (inset; 1 mM).

 

AMPA Receptor (GluA2) - Reproducible Responses

icon pap   GluA2 Stability PaPPort-a-Patch data and applications:
Cells were kindly provided by SB Drug Discovery.

GluA2 reproducibly recorded on the Port-a-Patch. L-glutamate was applied for 500 ms and this was repeated six times in the same cell showing reproducible responses.

AMPA Receptor (GluA2) - Fast Activation

GluR2 CRC current time course

icon pl   Patchliner data and applications:
Cells were kindly provided by University of Sussex.

Shown is concentration dependent activation of GluA2 receptors (known as AMPA receptors) by 10 μM, 30 μM, 100 μM, 300 µM and 1 mM Na-Glutamate from a GluA2 expressing HEK293 cell. The rising phase is enlarged in the right graph.

AMPA Receptor (GluA2) - Current Traces

icon sp96   SyncroPatch 384PE data and applications:
Cells were kindly provided by University of Sussex.

Using a stacked solutions approach and a fast pipetting speed shortens the solution exchange rate and minimizes the ligand exposure time. This procedure allows for reproducible recordings of fast desensitizing ligand-gated receptors such as glutamate receptors.
Here, repetitive activation of GluA2 receptors is shown. Receptors were activated with 100 µM Na-Glutamate for 3 times resulting in inward currents of similar peak amplitudes (A and B). The current onset time was approximately 10 ms (D). Panel C displays an example of a cumulative concentration response curve for Na-Glutamate (in mM: 0.1, 0.3 and 1).

Application Notes

Publications

2016 - Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module

icon sp96  SyncroPatch 384PE publication in Journal of Lab Automation (2016)

Authors: 
Obergrussberger A., Brüggemann A., Goetze T.A., Rapedius M., Haarmann C., Rinke I., Becker N., Oka T., Ohtsuki A., Stengel T., Vogel M., Steindl J., Mueller M., Stiehler J., George M., Fertig N.

Cookies make it easier for us to provide you with our services. With the usage of our services you permit us to use cookies.
More information Ok