05.10.2021 | Ionotropic Glutamate Receptors: Structure, Function and Dysfunction
Conference Venue: Virtual Event
Go to the Conference website here
Dr. Alison Obergrussberger (Nanion Technologies GmbH) will present the following poster:
"Speed and reliability matter: Fully automated patch clamp recordings of glutamate receptors"
When: 15:30 PM CET
The ionotropic glutamate receptors, N-Methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the mammalian CNS. The functional receptors exist as tetramers from a repertoire of four (GluA1 – GluA4) subunits for AMPA receptors or seven subunits for the NMDA receptor (GluN1, GluN2A-D and GluN3A-B2). AMPA receptors exist as homomers or heteromers whereas NMDA receptors are heterotetramers consisting of two GluN1 subunits and either two GluN2 subunits or a combination of GluN2 and GluN3 subunits.
We have used high and medium throughput automated patch clamp (APC) devices to record NMDA receptor subunit combinations GluN1 with either GluN2A, 2B, 2C or 2D and AMPA GluA2 receptors expressed in HEK cells. NMDA receptor-mediated responses were elicited using glutamate and glycine. Current amplitudes and desensitization kinetics were dependent on receptor subunit combination and different subunit combinations could be recorded simultaneously on a single 384-well chip so that these parameters could be directly compared within one experiment. Pharmacological agents were used to either enhance or block NMDA receptors-mediated responses with different subunit combinations.
AMPA receptors are fast activating and fast desensitizing, which can be challenging to record in patch clamp experiments involving cell lines overexpressing the channel. The presence of auxiliary subunits such as stargazin can also affect desensitization resulting in a sustained current component. We used GluA2 expressed in HEK cells on the SyncroPatch 384 and Patchliner and found that the speed of ligand application had profound effects on receptor activation and desensitization. Furthermore, responses to glutamate were reproducible and concentration response curves to glutamate were comparable across APC platforms. Using pharmacological agents we could either inhibit or enhance the glutamate elicited responses.
The use of high throughput APC enables the generation of hundreds of data points in a short space of time, different subunit combinations can be compared within a single experiment, and adjustment of the speed of ligand application ensures optimal parameters for recording ionotropic glutamate receptors.