| Assay Method Information | |
| | GABAA (α1β2γ2) Receptor Current using Patch-Clamp Technique |
| Description: | A whole-cell patch-clamp technique was used to study the allosteric regulation effect of the compound of the present invention on the GABAA (α1β2γ2) receptor.HEK293 cell lines stably expressing the GABAA (α1β2γ2) receptor were used. The GABAA (α1β2γ2) receptor gene information was as follows: GABA-α1: NM_000806; GABA-β2: NM_021911; GABA-γ2: NM_198904. The voltage stimulus of GABA receptor current recorded by a whole-cell patch-clamp technique was as follows: when whole-cell sealing was formed, the cell membrane voltage was clamped at −70 mV. The peak value of current was recorded after sequentially spraying test compounds from low concentration to high concentration and 100 μM GABA onto the cell surface in a Gap-free mode. The mode of administration of test compounds was as follows: for each concentration, the test compound was administered 1-2 times; the cells were washed with extracellular fluid for 1 min before detection was performed on the test compound at another concentration; and finally, 100 μM GABA was given as the control. The experimental data was collected by an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software. A microelectrode puller was used to pull capillary glass tubes into a recording electrode. A microelectrode manipulator was manipulated under an inverted microscope to contact the recording electrode with cells, and negative pressure suction was applied to form a GΩ seal. After the GΩ seal was formed, rapid capacitance compensation (pF) was conducted, and then negative pressure was continued to break cell membranes, forming a whole-cell recording mode. Then slow capacitance compensation was conducted, and the membrane capacitance (pF) and series resistance were recorded. |
| Affinity data for this assay | |
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