Plasmid construction, cell culture and transient transfection of CHO cells

The coding sequence for wild-type cASIC1a was sub-cloned into the pcDNA3.1/Zeo( + ) vector. All site-directed mutations were generated with overlap PCR and inserted into pcDNA3.1/Zeo(+). The mutants were sequenced to verify that no unwanted mutations had been introduced. Chinese hamster ovary (CHO) cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a 5% CO₂ incubator. The CHO cells were transferred to 24-well plates for transfection. When the CHO cells reached 90% confluence, they were transfected with 0.6 μg of plasmid encoding EGFP and 0.8 μg of plasmid encoding wild-type or mutant cASIC1a using Lipofectamine 2000 (Invitrogen, USA). After incubation for 5 h, the cells were transferred to poly-L-lysine (Sigma)-coated slides for culture for another 24–48 h in fresh medium. They were then used for the electrophysiological analysis.