12 mm round glass coverslips

HEK293T cells (ATCC) were transfected with rabbit TRPV2 cDNA mixed with EGFP with Lipofectamine 2000 (Invitrogen) at 6 μg of DNA in a 60 mm dish (1:1 ratio (w/w)). HEK293T cells, commonly used for functional characterization of TRP channels, were used for electrophysiology and calcium imaging. Cells tested negative for mycoplasma contamination. Cells were reseeded on 12-mm round glass coverslips (Warner Instruments) 1 d after transfection. Approximately 48 h after transfection, whole-cell recordings were performed on single isolated green cells identified under a fluorescence microscope at room temperature. Glass pipettes (Sutter Instrument Company) were prepared (3–4 MΩ) with a pipette puller P1000 (Sutter Instrument Company). Data were acquired with an Axopatch 200B amplifier controlled by a Clampex 10 via a Digidata 1440A data-acquisition system (Axon Instruments). Currents were sampled at a rate of 10 kHz and filtered at 3 kHz. The pipette solution contained 150 mM NaCl, 3 mM MgCl₂, 5 mM EGTA and 10 mM HEPES, adjusted to pH 7.2, and bath solution containing 150 mM NaCl, 6 mM CsCl, 1.5 mM CaCl₂, 1 mMgCl₂, 10 mM glucose and 10 mM HEPES, adjusted to pH 7.4.