Whole-Cell Patch-Clamp Recording

HEK293 cells that stably expressed AMPA-GluA2 receptors were seeded into the recording chamber 2 days before patch-clamp recordings, and the experiments were conducted in whole-cell configuration. Thin-walled borosilicate glass (Sutter Instrument, CA, United States) was pulled to make patch pipettes. The patch pipettes had open tips with resistance of 4–8 MΩ and were filled with internal electrode solution (mM): 135 CsCl (Sigma, Beijing, China), 10 CsF (Tocris, Bristol, United Kingdom), 10 HEPES (Amresco, United States), 5 Cs4BAPTA (Tocris, Bristol, United Kingdom), 1 MgCl₂, 0.5 CaCl₂, pH 7.2. The external bath solution consisted of (mM): 135 NaCl, 5 KCl, 10 HEPES, 1 MgCl₂, 1.8 CaCl₂, pH 7.35. The holding potential was set as -80 mV by an Axopatch-700B amplifier (Axon Instruments, CA, United States), and pClamp8 software (Axon Instruments, CA, United States) was used for data acquisition. Currents were recorded and digitized at 10 kHz. A fast-solution switch system was utilized for agonist (L-glutamate, Novabiochem, Darmstadt, Germany) application at varying concentrations, which allowed constant exchange of bath solutions to the cells, and stopped momentarily on the application of agonist or drugs. LCX001 and CX614 were dissolved in dimethyl sulfoxide (DMSO) before dilution with external bath solution (DMSO final concentration 0.5%). Dose-response curves were fitted to the Hill equation using Origin 9.0 software.