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.
Highlights
The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming ␣ subunits in Xenopus laevis oocytes
We demonstrate that Xenopus oocytes express relatives of mammalian MinK-related peptides (MiRPs) and that these endogenous xMiRPs can interact with injected mammalian Kv channel ␣ subunits
HERG, like KCNQ1, is a human cardiac Kv channel widely studied because of the role it plays in cardiac repolarization and the propensity of HERG to interact nonspecifically with a wide range of therapeutic agents, a propensity that contributes to acquired cardiac arrhythmia [21, 32]
Summary
Voltage-gated potassium channel; MiRP, MinK-related peptide; hMiRP, human MiRP; CHO, Chinese hamster ovary; RNAi, RNA interference; siRNA, short interfering RNA; TEVC, two-electrode voltage clamp; IKs, slow activating potassium current; IKr, rapidly activating potassium current; Ito, transient outward current; RT, reverse transcriptase; EST, expressed sequence tag; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Unit currents vary with the expression system or with the concentration of MiRP cRNA, even at levels where ␣-subunit saturation would be expected [6, 16, 17]. This finding suggests that these interactions depend on factors intrinsic to the expression system used. Resolving the true heteromultimeric identity of native ion channels and currents recorded in heterologous expression studies is important for our understanding of channel structure-function and physiology. We cloned a family of xMiRPs endogenously expressed in Xenopus oocytes and demonstrated that xMiRP2 interacts with overexpressed HERG to shape the functional attributes of HERG during oocyte expression studies
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