Ca2+ Imaging of TRPM3 Channels

Ca²⁺ imaging experiments were performed using an Olympus IX-51 inverted microscope equipped with a DeltaRAM excitation light source (Photon Technology International, PTI), as described earlier (Badheka et al., 2017 (link)). HEK293 cells were transfected with hTRPM3α2-GFP or its mutants using the Effectene reagent (Qiagen). Cells were loaded with 1 μM fura-2 AM (Invitrogen) for 40 min before the measurements at 37°C, and dual-excitation images at 340 and 380 nm excitation wavelengths were detected at 510 nm with a Roper Cool-Snap digital CCD camera. Measurements were conducted at room temperature in extracellular solution containing 137 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES and 10 mM glucose, pH 7.4. PregS, and primidone were applied with a gravity-driven whole chamber perfusion system. Temperature stimulation was performed using a custom-built system as described earlier (Badheka et al., 2017 (link)) by pushing bath solution through a spiral tubing immersed in hot water using a 60 ml syringe while monitoring the temperature of the perfusion chamber using a CL-100 Warner Instruments temperature controller. The analogue signal from the CL-100 unit was fed into the Digidata digitizer and the temperature curve was collected in Clampex. Data analysis was performed using the Image Master 5 software (PTI).