Kir2.x inward rectifier potassium channels are differentially regulated by adrenergic alpha1A receptors.
Kir2.x inward rectifier potassium channels are differentially regulated by adrenergic alpha1A receptors.
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DOI：
10.1016/j.yjmcc.2007.10.008
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发表时间：
2008
影响因子：
5
通讯作者：
E. Zitron;Myriam Günth;D. Scherer;Claudia Kiesecker;M. Kulzer;Ramona Bloehs;E. Scholz;Dierk Thomas;Christian Weidenhammer;S. Kathöfer;A. Bauer;H. Katus;C. Karle
中科院分区：
文献类型：
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作者：
E. Zitron;Myriam Günth;D. Scherer;Claudia Kiesecker;M. Kulzer;Ramona Bloehs;E. Scholz;Dierk Thomas;Christian Weidenhammer;S. Kathöfer;A. Bauer;H. Katus;C. Karle
在心肌细胞中观察到肾上腺素能 α1 受体对 IK1 电流的抑制，并且在动物模型中与心律失常发生有关。该调节中隐含了 PKC 依赖性和 PKC 独立途径。然而，迄今为止，其潜在的分子机制尚未阐明。天然 IK1 电流的分子基础主要由蛋白激酶差异调节的 Kir2.1 (KCNJ2)、Kir2.2 (KCNJ12) 和 Kir2.3 (KCNJ4) 通道形成。因此，我们试图研究这些不同的 Kir2.x 通道亚基在此调节中的作用，并确定所涉及的主要信号传导途径。肾上腺素 α1A 受体（主要的心脏亚型）与爪蟾卵母细胞中克隆的 Kir2.1、Kir2.2 和 Kir2.3 通道共表达，并使用双微电极电压钳进行电生理实验。使用全细胞膜片钳技术测量离体大鼠心室心肌细胞中的天然 IK1 电流。去氧肾上腺素激活共表达的肾上腺素能 α1A 受体，在 Kir2.x 通道中诱导差异效应。 Kir2.1 通道中未发现任何影响。然而，在 Kir2.2 通道中观察到明显的抑制作用。这种调节不会被 PKC、CamKII 和 PKA 抑制剂（白屈菜红碱、KN-93、KT-5720）减弱，并且缺乏 PKC 和 PKA 功能性磷酸化位点的突变 Kir2.2 通道表现出与 Kir2.2 野生型通道相同的效果。相比之下，该调节可被一般酪氨酸激酶抑制剂金雀异黄素和 src 酪氨酸激酶抑制剂 PP2 抑制，这表明 src 激酶的重要作用。这一发现在大鼠心室心肌细胞中得到了验证，其中 PP2 的共同应用强烈减弱了肾上腺素能 α1 受体对 IK1 电流的抑制调节。无活性类似物 PP3 作为 PP2 的阴性对照进行测试，并且没有重现 PP2 的作用。在Kir2.3通道中，观察到α1A受体激活的显着抑制作用。这种调节可以通过白屈菜红碱或 Ro-32-0432 抑制 PKC 来减弱，但不能通过金雀异黄素抑制酪氨酸激酶来减弱。 总之，在分子水平上，肾上腺素能 α1A 受体对 IK1 电流的抑制调节可能基于对 Kir2.2 和 Kir2.3 通道的影响。 Kir2.2 通过独立于蛋白激酶 C 的 src 酪氨酸激酶途径进行调节，而 Kir2.3 受到蛋白激酶 C 依赖性途径的抑制。 Src 酪氨酸激酶途径对于肾上腺素能 α1 受体抑制天然 IK1 电流至关重要。这种调节可能有助于肾上腺素刺激下的心律失常发生。
Inhibition of IK1currents by adrenergic α1receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native IK1current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved.Adrenergic α1Areceptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native IK1currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes.Activation of co-expressed adrenergic α1Areceptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of IK1current by adrenergic α1receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of α1Areceptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein.In summary, on the molecular level the inhibitory regulation of IK1currents by adrenergic α1Areceptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native IK1current by adrenergic α1receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation.