Channel subunit cRNA preparation and Xenopus laevis oocyte injection

As previously described (9), we generated cRNA transcripts encoding human KCNQ4 and KCNQ5 by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA sub-cloned into plasmids incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability. We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US) with KCNQ cRNAs (5–10 ng). We incubated the oocytes at 16 °C in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 3–4 days prior to two-electrode voltage-clamp (TEVC) recording.