Isosakuranetin

Standard whole-cell patch-clamp recordings were performed with an EPC-10 amplifier and the PatchMasterPro Software (HEKA Elektronik, Lambrecht, Germany). Current measurements were performed at a sampling rate of 20 kHz and currents were digitally filtered at 2.9 kHz. In all measurements, 70% of the series resistance was compensated. The standard internal solution contained (in mM): 100 CsAsp, 45 CsCl, 10 EGTA, 10 HEPES, 1 MgCl₂ (pH 7.2 with CsOH) or 140 potassium gluconate, 5 EGTA, 1 MgCl₂, 10 HEPES and 2 NaATP (pH 7.3 with CsOH) for the measurements of G_βγ mediated inhibition and the standard extracellular solution contained (in mM): 150 NaCl, 1 MgCl₂, 10 HEPES (pH 7.4 with NaOH). The standard patch pipette resistance was between 2 MΩ and 4 MΩ when filled with pipette solution. In experiments allowing Ca²⁺-dependent desensitization of TRPM3, Mg²⁺ was replaced by Ca²⁺ in the extracellular solution, and Cs⁺ was replaced by Na⁺ in the pipette solution. Pregnenolone sulfate, clotrimazole primidone and [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate salt (DAMGO) were obtained from Sigma-Aldrich and isosakuranetin was obtained from Carl Roth. The chemical ligands were dissolved in bath solution from a stock diluted in DMSO.

Changes in intracellular calcium concentration were monitored using ratiometric Fura-2-based fluorimetry. Cells were loaded with 2 µM Fura-2-acetoxymethyl ester (Alexis Biochemicals) for 30 min at 37°C. Fluorescence was measured during alternating illumination at 340 and 380 nm using Eclipse Ti (Nikon) fluorescence microscopy system, and absolute calcium concentration was calculated from the ratio of the fluorescence signals at these two wavelengths (R = F₃₄₀/F₃₈₀) as [Ca²⁺]=K_m × (R-R_min)/(R_max-R), where K_m, R_min and R_max were estimated from in vitro calibration experiments with known calcium concentrations. The bath solution contained (in mM) 138 NaCl, 5.4 KCl, 2 CaCl₂, 2 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4. Pregnenolone sulfate, clotrimazole and primidone were obtained from Sigma-Aldrich, isosakuranetin was obtained from Carl Roth. The chemical ligands were dissolved in bath solution from a stock diluted in DMSO.