Ml204

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) and Carl Roth (Karlsruhe, Germany) (unless stated otherwise). Carbachol was purchased from Alfa Aesar (Kandel, Germany), Clemizole hydrochloride and ML204 were obtained from Tocris (Wiesbaden-Nordenstadt, Germany), Pico145 was given by Dr. Robin Bon (University of Leeds, Leeds, UK). Carbachol stock solution was prepared in water. Clemizole hydrochloride, ML204 and Pico 145 were diluted in DMSO and, to prevent degradation, they were stored at −20 °C and thawed right before use. DMSO stock solutions were diluted to less than 1:1000 in nACSF.
The antibodies used in the patch clamp experiments were the anti-TRPC4 from Neuromab (USA) and the anti-TRPC5 from Almone Labs (Israel). Both antibodies targeted an epitope in the intracellular part of the channels. The anti-TRPC4 antibodies targeted the C-terminal tail of the TRPC4 channel (Epitope: 930-947), while the anti-TRPC5 antibodies targeted the C-terminal tail of the TRPC5 channel (Epitope: 959-973). Inactivation of the antibodies was carried out by incubating the antibodies at 90 °C for 10 min.

Radius^TM 96‐well cell migration assay (Cell Biolabs, San Diego, CA, USA) was used for quantification of cell migration according to the manufacturer's protocol. Culture medium with different pH values was prepared by buffering with different amounts of bicarbonate (44 mm for pH 7.7, 22 mm for pH 7.4, 5.5 mm for pH 6.8, 2 mm for 6.4 mm). Experiments were done in culture medium under control conditions (nothing added), with different pH, or in the additional presence of drugs under investigation (alone or in combination) (pluronic acid (0.01% v/v), DMSO (0.1% v/v), (−)EA (PhytoLab; 10 μm), ML204 (10 μm; Tocris Bioscience, Bristol, UK) clemizole hydrochlorid (Tocris; 10 μm) for 6 h at 37°C under 5% CO₂. Assays were performed in triplicates, the average of which was used and represents the result of one migration assay. Usually, at least three distinct and independent migration assays were carried out for each condition. Plates were photographed at 0 h and 6 or 12 h at the identical location of the initial image. Quantifications of migration ability were performed with ImageJ software.

ML204 (4-Methyl-2-(1-piperidinyl)-quinoline), α,β-Methylene ATP and clemizole hydrochloride (Tocris), neostigmine & carbachol (Sigma).

(-)-Englerin A (EA) (2 or 4 mg/kg; ITW Reagents, Germany) or vehicle (0.5% EtOH, 1% PEG300, 0.5% Cremaphor EL in saline, i.p.) was given 30 min before carrageenan injection. In some experiments, mice were treated with the TRPC4/5 antagonist ML204 (2 mg/kg; Tocris, Bristol, U.K.) or vehicle (2% DMSO in saline, i.p.) 1 h prior to the induction of paw oedema.