Two electrode voltage clamp experiments

Xenopus laevis oocytes were prepared as described earlier (Badheka et al., 2015). All animal procedures were approved by the Institutional Animal Care and Use Committee at Rutgers New Jersey Medical School. In brief, frogs were anesthetized in 0.25% ethyl 3-aminobenzoate methanesulfonate solution (MS222; Sigma-Aldrich); bags of ovaries were removed surgically from the anesthetized frogs. Individual oocytes were obtained by overnight digestion at 16°C in 0.2–0.3 mg/ml type 1A collagenase (Sigma-Aldrich), dissolved in a solution containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 μM HEPES, pH 7.4 (OR2 solution). The next day the collagenase containing solution was discarded and the oocytes were washed multiple times with OR2 solution. The oocytes were maintained in OR2 solution supplemented with 1.8 mM CaCl₂, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 16°C. cRNA was transcribed from the linearized human TRPM3 (hTRPM3) cDNA clone (Grimm et al., 2003), or its mutants in the pGEMSH vector using the mMessage mMachine kit (Thermo Fisher Scientific). cRNA (40 ng) was microinjected into individual oocytes, using a nanoliter-injector system (Warner Instruments). For combined injection of wild-type and mutant TRPM3 for Figures 4E–G, 40 μg total cRNA was injected in a 1:1 ratio. The V990M and P1090Q mutants were generated using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). For the GPCR regulation of these mutants, we injected cRNA of human M2 muscarinic receptors together with TRPM3 or mutants at 1:1 ratio. Oocytes were used for electrophysiological measurements 48–72 hr after microinjection. The hTRPM3₁₃₂₅ clone in a mammalian expression vector was provided by C. Harteneck (Eberhard Karls University Tübingen, Tübingen, Germany), and it was subcloned into the pGEMSH oocyte vector using standard molecular biology techniques.

Two electrode voltage clamp experiments were performed as described (Badheka et al., 2015). In brief, oocytes were placed in extracellular solution (97 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 μM HEPES, pH 7.4), and currents were recorded with thin-wall inner filament– containing glass pipettes (World Precision Instruments) filled with 3 M KCl in 1% agarose. Currents were measured with a ramp protocol from −100 to 100 mV once every 0.5 s with a GeneClamp 500B amplifier and analyzed with the pClamp 9.0 software (Molecular Devices). PregS, ACh and primidone were applied with a gravity driven whole chamber perfusion system. Temperature stimulation was performed the same way as for the whole cell patch clamp and Ca²⁺ imaging experiments in HEK293 cells.