T7 polymerase mmessage mmachine kit

cRNA transcripts encoding hKv4.3L, hKv4.3S and hKChIP2b were generated by in vitro transcription (T7 polymerase mMessage mMachine kit, Thermo Fisher Scientific), after vector linearization, from cDNA sub-cloned into plasmids (a kind gift of Dr. Steve A. N. Goldstein, Brandeis University, Waltham, MA) incorporating X. laevis β-globin 5′ and 3′ UTRs flanking the coding region to enhance translation and cRNA stability. T504A and T504D Kv4.3L mutants were made using a QuikChange Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA) per the manufacturer's protocol and confirmed by sequencing. Human KCNE2 and KCNE4L were also transcribed from cDNA templates incorporating X. laevis β-globin 5′ and 3′ UTRs. cRNA was quantified by spectrophotometry. Defolliculated stage V and VI X. laevis oocytes (Ecocyte Bioscience, Austin, TX) were injected with one, two or three of the subunit cRNAs as follows: 1.5 ng of Kv4.3L or Kv4.3S; with or without 5 ng of KChIP2b, with or without 5 ng of KCNEx. Oocytes were incubated at 16°C in SBB solution (Ecocyte) containing penicillin and streptomycin, with daily washing, for 2–3 days prior to two-electrode voltage-clamp (TEVC) recording.

We generated cRNA transcripts encoding human KCNQ1, KCNQ2, KCNQ3, KCNQ4, KCNQ5 or GLRA1 (NM_001146040) (GenScript, Piscataway, NJ, USA) by in vitro transcription using the T7 polymerase 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 quantified cRNA by spectrophotometry. We generated mutant KCNQ2 and KCNQ3 cDNAs by site-directed mutagenesis using a QuikChange kit (Stratagene, San Diego, CA) and prepared the corresponding cRNAs as above. We injected defolliculated stage V and VI Xenopus laevis oocytes (Ecocyte Bioscience, Austin, TX and Xenoocyte, Dexter, MI) with KCNQ channel α subunit (5-20 ng) or GLRA1 (20 ng) cRNAs. We incubated the oocytes at 16 °C in Barth’s saline solution (Ecocyte Bioscience) containing penicillin and streptomycin, with daily washing, for 2–5 days prior to two-electrode voltage-clamp (TEVC) recording.

cRNA transcripts encoding human KCNQ1, KCNQ2, KCNQ3, KCNQ4, KCNQ5, KCNA1, or KCNE1 were generated by in vitro transcription using the T7 polymerase mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from complementary DNA (cDNA) sub-cloned into plasmids incorporating Xenopus laevis β-globin 5′ and 3′ untranslated regions flanking the coding region to enhance translation and cRNA stability. cRNA was quantified by spectrophotometry. Mutant KCNQ3 cDNAs were generated by site-directed mutagenesis using a QuikChange kit according to manufacturer’s protocol (Stratagene, San Diego, CA) and corresponding cRNAs prepared as above. Defolliculated stage V and VI Xenopus laevis oocytes (Ecocyte Bioscience, Austin, TX) were injected with Kv channel α subunit cRNAs (5–10 ng). Oocytes were incubated at 16 °C in Barth’s saline solution (Ecocyte) containing penicillin and streptomycin, with daily washing, for 3–5 days prior to TEVC recording or ³H-GABA binding assays.

cRNA transcripts encoding hKCNE3L and hKCNE4L were generated by in vitro transcription (T7 polymerase mMessage mMachine kit, Thermo Fisher Scientific) from synthetic genes sub-cloned into pCDNA3.1+ with Xenopus laevis β-globin 5′ and 3′ UTRs flanking the coding region to enhance translation and cRNA stability, after vector linearization. hKCNE3S and hKCNE4S cRNAs were similarly generated, after using site-directed mutagenesis (Agilent, Santa Clara, CA), to disrupt the exon 1 start site on the cDNAs of their long counterparts to facilitate direct comparisons of short versus long isoforms in otherwise identical constructs. Human Kv4.2, Kv4.3 (full-length) and KChIP2 were similarly transcribed from cDNA templates also incorporating Xenopus laevis β-globin 5′ and 3′ UTRs (a kind gift of Dr. Steve A. N. Goldstein), as were human HCN1, hERG and Kv1.1. cRNA was quantified by spectrophotometry. Defolliculated stage V and VI Xenopus laevis oocytes (Ecocyte Bioscience, Austin, TX) were injected with one, two or three of the subunit cRNAs (5 ng of KCNE subunits, 4 ng hERG, 10 ng HCN1, 1–5 ng KChIP2, 0.1 ng Kv1.1, 1 ng Kv4.2, 1–5 ng Kv4.3 per oocyte). Oocytes were incubated at 16 °C in SBB solution (Ecocyte) containing penicillin and streptomycin, with daily washing, for 2–3 days before two-electrode voltage-clamp (TEVC) recording.