title: RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies.
abstract: The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function.
authors.affiliation: Division of Cardiology, Department of Medicine and Pharmacology, Graduate Program of Neuroscience, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: Arun
authors.initials: A
authors.lastname: Anantharam
authors.forename: Anthony
authors.initials: A
authors.lastname: Lewis
authors.forename: Gianina
authors.initials: G
authors.lastname: Panaghie
authors.forename: Earl
authors.initials: E
authors.lastname: Gordon
authors.forename: Zoe A
authors.initials: ZA
authors.lastname: McCrossan
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lerner
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Abbott
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D012313:RNA / Q000494:pharmacology; D034622:RNA Interference / Q000502:physiology*; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014982:Xenopus laevis
chemical_list: D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C418492:KCNE3 protein, human; C000606913:KCNH2 protein, human; C092509:KCNH6 protein, human; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; C059983:potassium channel protein I(sk); D012313:RNA