Heterologous Expression of KCNE and KCNQ Channels

As previously described^{7 (link)}, we generated cRNA transcripts encoding human KCNE1, KCNE3, KCNQ1, KCNQ2, KCNQ3, KCNQ4 or 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 generated mutant KCNQ1, KCNQ2 and KCNQ3 cDNAs by site-directed mutagenesis with a QuikChange kit (Stratagene, San Diego, CA) and prepared the cRNAs as above. We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US and NASCO, Fort Atkinson, WI, US) with KCNE and/or KCNQ cRNAs (2–20 ng). We incubated the oocytes at 16 °C in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 2–4 days prior to TEVC recording.