Cell culture and transfection of HEK293 cells

For whole-cell electrophysiology, single-channel electrophysiology, and immunohistochemistry, HEK293 cells (ATCC; mycoplasma tested negative) were cultured on glass coverslips in 10 cm dishes and transfected by a calcium phosphate method (Peterson et al., 1999) with the following amounts of DNA: 3 µg of SV40 T antigen to enhance expression, 2–8 µg of α₁-subunit of Ca²⁺ or Na⁺ channel depending on expression, 8 µg from rat β_2A (Perez-Reyes et al., 1992) ({"type":"entrez-nucleotide","attrs":{"text":"M80545","term_id":"203223","term_text":"M80545"}}M80545), 8 µg from rat α₂δ (Tomlinson et al., 1993) (NM012919.2), and 8 µg of the stac1, stac2, or stac3 variants indicated.

For FRET two-hybrid experiments, cells were cultured on glass-bottom dishes and transfected with a standard polyethylenimine protocol (Lambert et al., 1996). Epifluorescence measurements were recorded 1–2 days after transfection.