D ala2

All the drugs used in the present study, including morphine, D-Ala², MePhe⁴, Gly⁵-ol enkephalin (DAMGO), naloxone, Gö6983, dopamine and phenylephrine (PE) were purchased from Sigma-Aldrich. For acute drug treatment, cells were seeded onto six-well plates for 24 h at densities of 5×10⁵ cells/well (CHO/μ cells and HEK 293 cells) or 1×10⁶ cells/well (SH-SY5Y cells), and were serum-starved overnight upon reaching 80% confluence prior to stimulation by different concentration of drugs or vehicles. For morphine chronic treatment, cells were seeded onto six-well plates for 12 h prior to treatment, at densities of 2×10⁵ cells/well (CHO/μ cells) or 5×10⁵ cells/well (SH-SY5Y cells), and then treated with morphine or vehicle for 36 h.

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.