Cell Culture and Transfection

Human embryonic kidney (HEK293T) cells containing the SV40 Large T-antigen were cultured at 37°C, 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen Life Technologies, Australia), supplemented with 10% fetal bovine serum (FBS, Bovigen, Australia), 1% GlutaMAX and penicillin-streptomycin (Invitrogen, Australia). HEK293 cells were then transiently co-transfected with the different voltage-gated calcium channel isoforms and green fluorescent protein (GFP) for visualization, using the calcium phosphate method. cDNAs encoding mCa_v1.2 (a gift from Dr. D. Lipscombe; Addgene plasmid #26572) (Helton et al., 2005), rCa_v1.3 (a gift from Dr. D. Lipscombe; Addgene plasmid # 49,333) (Xu and Lipscombe, 2001), hCa_v2.1 (a gift from Dr. J. Striessnig), rCa_v2.2 (a gift from Dr. D. Lipscombe), hCa_v2.2 (a gift from Dr. D. Yue), hCa_v2.3 (purchased from GenScript United States Inc.) were co-transfected with β₃, α₂δ₁ and GFP. After transfection, cells were plated on 12 mm cover glass and transferred to a 30°C incubator for 1–3 days.