12 mm coverslips

HEK293T cells that were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich or Wako) supplemented with 10% fetal bovine serum (FBS) (Biowest) at 37°C under 5% CO₂ in air were seeded on a 35-mm dish (Iwaki) at 5 × 10⁵ cells/well, grown overnight, and transfected with pRc/CMV-mB2R (100 (link)), pcDNA-hTRPV1 (100 (link)), pcDNA-hTRPV1_S117A/T371A, pCMV-SPORT6-hADRA2A (DNAFORM, ID 6198830), and/or pGreen-Lantern 1 (101 (link)), which is a humanized green fluorescent protein (GFP) expression vector, using Lipofectamine reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. pcDNA-hTRPV1_S117A/T371A was generated from pcDNA-hTRPV1 by site-directed mutagenesis to replace Ser¹¹⁷ and Thr³⁷¹ with Ala by using a PrimeSTAR Mutagenesis Basal kit (TaKaRa Bio) with the primer sets S117A-S and S117A-AS, and T371A-S and T371-AS (Table S1). After incubation for 3 to 4 h, the cells were reseeded on 12-mm coverslips (Matsunami Glass), further incubated for 2 days, and subjected to whole-cell patch-clamp recordings.

The human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco’s modified Eagle Medium (Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin-streptomycin (50 mg/mL and 50 units/mL, respectively, Gibco) and GlutaMAX (2 mM, Gibco). For transient transfection of HEK293 cells, 1 μg of plasmid DNA in pcDNA3.1 (+) and 0.1 μg pGreen-Lantern 1 vector were transfected into HEK293 cells using Lipofectamine reagent and Plus reagent (Invitrogen, Carlsbad, CA, USA). In the case of transfection for calcium-imaging, 0.1 μg pCMV-DsRed vector was transfected instead of pGreen-Lantern 1 vector. All these components were dissolved in OPTI-MEM medium (1X, Gibco). After incubation for 3–4 h, HEK293 cells were reseeded on 12-mm cover slips (Matsunami, Tokyo, Japan) and further incubated at 33 °C in 5% CO₂.