Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel
Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel
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DOI：
10.1093/cvr/cvn052
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发表时间：
2008-06-01
影响因子：
10.8
通讯作者：
Kuroda, Shun'ichi
中科院分区：
文献类型：
--
作者：
Maturana, Andres D.;Waelchli, Sebastien;Kuroda, Shun'ichi
在心肌细胞中，蛋白激酶D1（PKD 1）在应激反应中起着重要作用。从酵母双杂交试验，我们已经确定了谜同源物1（ENH 1）作为PKD 1的新的结合伴侣。由于在神经元中，与蛋白激酶C β相关的ENH 1被证明可调节N型钙通道的活性，而心脏L型电压门控钙通道的成孔亚基α 1C具有PKD 1的潜在磷酸化位点，我们在此研究了ENH 1和PKD 1在调节心脏L型电压门控钙通道中的可能作用。通过酵母双杂交筛选相互作用蛋白。用免疫共沉淀法检测了从新生大鼠心室分离的心肌细胞中的体内蛋白质相互作用。通过使用腺病毒将小干扰RNA和PKD 1的显性失活突变体递送到心肌细胞中。用膜片钳技术测定钙电流。ENH 1和PKD 1都与心肌细胞中的α 1C相互作用。这种相互作用在刺激时增加。ENH 1的沉默阻止PKD 1与α 1C的结合。此外，PKD 1的显性负突变体或ENH 1的沉默抑制α-肾上腺素能诱导的L-型钙电流的增加。结论我们发现了一个新的结合伴侣，ENH 1，和一个新的目标，α 1C，PKD 1在新生大鼠心肌细胞。我们提出了一个模型，ENH 1支架PKD 1的α 1C，以形成一个信号复合物，调节心脏L型电压门控Ca（2+）通道的活性。
Aims In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase C epsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha 1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel.Methods and results PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiornyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha 1C in cardiornyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha 1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents.Conclusion We found a new binding partner, ENH1, and a new target, alpha 1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha 1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.