RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies
RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies
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DOI：
10.1074/jbc.m212751200
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发表时间：
2003-04-04
影响因子：
4.8
通讯作者：
Abbott, GW
中科院分区：
文献类型：
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作者：
Anantharam, A;Lewis, A;Abbott, GW
大多数离子通道的生理特性是通过非洲爪蟾卵母细胞中成孔α亚基的功能表达通过实验来定义的。在这里，我们克隆了非洲爪蟾 KCNE 基因家族，该基因编码 MinK 相关肽 K+ 通道 β 亚基 (xMiRP)，并证明了它们在卵母细胞中的组成型表达。 xMiRP2 的电生理学分析表明，当过度表达时，该基因通过抑制 HERG 电流并消除 KCNQ1 激活的电压依赖性来调节人心脏 K+ 通道亚基 HERG（人 ether-ago-go 相关基因）和 KCNQ1。接下来评估 xMiRP2 内源水平对卵母细胞研究中过度表达的哺乳动物 K+ 通道的生物物理属性的影响。注射 xMiRP2 序列特异性短干扰 RNA (siRNA) 寡核苷酸可将内源性 xMiRP2 表达降低 5 倍，而对照 siRNA 寡核苷酸则没有效果，这表明 RNA 干扰技术在非洲爪蟾卵母细胞中的有效性。使用异源表达的 HERG 通道的电生理学分析来测试内源性 xMiRP2 沉默的功能效应。 RNA 干扰介导的内源性 xMiRP2 表达减少使宏观 HERG 电流增加了 10 倍，具体取决于 HERG cRNA 浓度。人 MiRP1 (hMiRP1)/HERG 相互作用的功能效应也受到内源性 xMiRP2 的影响。在高 HERG 通道密度下，内源性 xMiRP2 的影响最小，hMiRP1 降低了 HERG 电流。在低 HERG 电流密度下，hMiRPI 反而上调 HERG 电流，这一结果与 hMiRP1 拯救 HERG 免受内源性 xMiRP2 抑制的结果一致。因此，内源性非洲爪蟾 MiRP 亚基有助于卵母细胞表达研究中 K+ 通道（如 HERG）的基线特性，这可以解释 K+ 通道功能中表达水平和表达系统依赖性的变化。
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 a subunits HERG (human ether-ago-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, hMiRPI 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 baseline properties of K+ channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K+ channel function.