T7 or sp6 mmessage mmachine transcription kit

For the expression of Na_V channels, including hNa_V1.1, rNa_V1.2, rNa_V1.3, rNa_V1.4, hNa_V1.5, mNa_V1.6, rNa_V1.7, rNa_V1.8, together with the auxiliary subunits rβ1 and hβ1, in Xenopus oocytes, the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion®, Carlsbad, California, USA). Stage V–VI Xenopus laevis oocytes were isolated by partial ovariectomy. The animals were anesthetized by a 15 min submersion in 0.1% tricaine methane sulfonate (Sigma®) solution (pH 7.0). Isolated oocytes were defolliculated with 1.5 mg/mL collagenase. Defolliculated oocytes were injected with 50 nL of cRNA at a concentration of 1 ng/nL using a micro-injector (Drummond Scientific®, Broomall, Pennsylvania, USA). The oocytes were incubated in a solution containing (in mM): NaCl, 96; KCl, 2; CaCl₂, 1.8; MgCl₂, 2 and HEPES, 5, at pH 7.4, supplemented with 50 mg/L gentamycin sulfate. The use of the frogs was in accordance with license number LA1210239 of the Laboratory of Toxicology & Pharmacology, University of Leuven. All animal care and experimental procedures agreed with the guidelines of ‘European convention for the protection of vertebrate animals used for experimental and other scientific purposes’ (Strasbourg, 18.III.1986).