Fura 2

The transfected HEK293 cells were seeded on glass coverslips (Matsunami) coated with poly-L-lysine solution (Sigma) and incubated for 4 h at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The calcium indicator Fura-2 AM (Dojindo) was loaded to the cells at 5 μM for 20–30 min. The imaging experiment was carried out in HBS buffer (20 mM HEPES pH 7.4, 107 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 2 mM CaCl₂, 11.5 mM glucose). The fluorescence images and the Fura-2 ratio were measured using a fluorescence microscope (IX71, Olympus) equipped with a complementary metal-oxide semiconductor (CMOS) camera (ORCA-flash 4.0, Hamamatsu Photonics) under xenon-lamp illumination, and analyzed with a video imaging system (AQUACOSMOS, Hamamatsu Photonics) following the manufacture’s instruction. In imaging experiments, three different HEK293 cells transfected with one of the mGlu1 mutants were co-cultured on a glass coverslip, and each mutant was visually distinguished by the transfection markers. These three different cells were assayed simultaneously. The Δratio value was defined as the difference between the maximum ratio value after adding the reagent (metal ion or complex, or glutamate) and the average ratio before adding the reagent. The Δratio was fitted with KaleidaGraph to calculate the EC₅₀ value using the equation: a + (b-a)/(1+(x/c)^d).

Intracellular Ca²⁺ levels of OSNs were monitored using Ca²⁺ sensitive dye Fura-2 as described in our previous publication (Ogura et al., 1997 (link), 2010 (link), 2011 (link)). Briefly, cells were loaded with 2 μM Fura-2 AM (Molecular Probes) for 20–25 min. A pair of Fura-2 fluorescence images were captured every 3 s at 340 and 380 nm excitation lights using an inverted microscope (Olympus IX71) equipped with a UAPO/340 40x objective lens, a Hamamatsu CCD camera, a Sutter LS Xenon light source/filter changer controlled by Imaging Workbench software version 6 (INDEC BioSystems, Santa Clara, CA, USA). We measured Ca²⁺ levels as ratio of fluorescence values from 340 nm excitation/380 nm excitation light images. A change in the intracellular Ca²⁺ levels (ratio of F340/F380) is considered to be a stimulus-induced response if the peak value of the change during stimulation was greater than 5% of the resting level and within 30 s after stimulation, The resting Ca²⁺ level was obtained by averaging 10 data points (3 s each) before applying the stimulus in each cell tested or using the value of the first point before an assumed response if the baseline was stable. In figures containing Ca²⁺ traces, the vertical scale bar stands for a 50% change in the Ca²⁺ level measured from the resting Ca²⁺ level of the recorded region of the same cells.

Intracellular Ca²⁺ concentration measurements were obtained from PTC of WT and TRPC6^-/- mice preloaded with the Ca²⁺-sensitive fluorescent dye Fura2-AM (Invitrogen, F1201, USA). As described in He et al.^{41 (link)}, PNAS 2017, briefly, the cells were loaded with 3 μM Fura2-AM in DMEM/F12 1:1 medium for 50 min at room temperature. Then the cells were washed 3 times with HBSS (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, and 1 mM MgCl₂, pH 7.4) medium with 2 mM Ca²⁺ and incubated at room temperature for another 10 min. The coverslips were mounted onto the platform of an inverted epifluorescence microscope. To measure Thapsigargin (Tg, Invitrogen, T7459, USA)-evoked Ca²⁺ entry, cells were bathed in sequence with 50 μM EGTA in HBSS for 3 min, 50 μM EGTA and 2 μM Tg in HBSS for 6 min, and 2 mM Ca²⁺ plus 2 μM Tg in HBSS for 6 min, as shown in the figures. Ca²⁺ entry was also assessed in the absence and presence of the TRPC inhibitor SAR7334. Cytosolic Ca²⁺ was monitored with an Olympus IX51 inverted fluorescence microscope and SlideBook software, using excitation wavelengths of 340 and 380 nm to detect Fura-2/Fura2-Ca²⁺ fluorescence emissions at 510 nm.