PKA and PKC partially rescue long QT type 1 phenotype by restoring channel-PIP2 interactions
PKA and PKC partially rescue long QT type 1 phenotype by restoring channel-PIP2 interactions
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DOI：
10.4161/chan.4.1.10227
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发表时间：
2010-01-01
期刊：
影响因子：
3.3
通讯作者：
Lopes, Coeli M. B.
中科院分区：
文献类型：
--
作者：
Matavel, Alessandra;Medei, Emiliano;Lopes, Coeli M. B.
长QT综合征可导致尖端扭转型室性心动过速心律失常、心室颤动和猝死。长QT综合征最常见的遗传形式，LQT 1，是由于钾通道基因KCNQ 1的突变，KCNQ 1形成了主要的复极化心脏K+通道之一，IKs。IKs已被证明由β-肾上腺素能受体（通过蛋白激酶a（pKa））和Gq蛋白偶联受体（GqpCR）（通过蛋白激酶C（pKC）和磷脂酰肌醇4，5-二磷酸（PIP2））调节。这些调节途径被证明是相互干扰的，PKa磷酸化增加了IKs对PIP2的表观亲和力。在这里，我们研究了假定的PIP2-KCNQ1相互作用位点中的LQT1突变对IKs通过pKa和GqpCR调节的影响。检测了LQT 1突变对IKs调节的影响，这些突变位于KCNQ 1亚基的四个不同胞质结构域：R174 C（S2-S3）、R243 C（S4-S5）、R366 Q（近端c-末端）和R555 C（远端c-末端）中的保守带正电荷氨基酸。IKs（近端和远端）C末端的突变增强了通道对膜PIP2水平变化的敏感性，这与表观通道-PIP2亲和力的降低一致。这些突变体通道对受体介导的PIP2耗尽引起的抑制更敏感，对通过磷脂酰肌醇-4-磷酸-5-激酶（PI5-激酶）过表达刺激PIP2产生更敏感。此外，C-末端突变体表现出通过pKa的增强调节。另一方面，对于两个胞质环突变，观察到pKa激活受损。突变对通道pKC刺激的影响与对pKa刺激的影响一致，表明两种调节输入同样受到突变的影响。我们测试了PKC介导的IKs激活是否类似于PKA介导的激活，可以调节对PIP2的通道反应。pKC激活后，通道对膜PIP2水平的变化不太敏感，这与通道-PIP2亲和力的增加一致。PKC激活通道对脂质激酶抑制剂渥曼青霉素阻断PIP2合成所引起的抑制不太敏感，对PIP2产生的刺激不太敏感。我们的数据表明，通过pKa和pKC的刺激可以通过加强通道与PIP2的相互作用来部分拯救对PIP2反应减弱的LQT 1突变体通道。
Long-QT syndrome causes torsade de pointes arrhythmia, ventricular fibrillation, and sudden death. The most commonly inherited form of long-QT syndrome, LQT1, is due to mutations on the potassium channel gene KCNQ1, which forms one of the main repolarizing cardiac K+ channels, IKs. IKs has been shown to be regulated by both beta-adrenergic receptors, via protein kinase a (pKa), and by Gq protein coupled receptors (GqpCR), via protein kinase C (pKC) and phosphatidylinositol 4,5-bisphosphate (PIP2). These regulatory pathways were shown to crosstalk, with pKa phosphorylation increasing the apparent affinity of IKs to PIP2. here we study the effects of LQT1 mutations in putative PIP2-KCNQ1 interaction sites on regulation of IKs by pKa and GqpCR. The effect of the LQT1 mutations on IKs regulation was tested for mutations in conserved, positively charged amino acids, located in four distinct cytoplamic domains of the KCNQ1 subunit: R174C (S2-S3), R243C (S4-S5), R366Q (proximal c-terminus) and R555C (distal c-terminus). Mutations in the c-terminus of IKs (both proximal and distal) enhanced channel sensitivity to changes in membrane PIP2 levels, consistent with a decrease in apparent channel-PIP2 affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP2-depletion and more sensitive to stimulation of PIP2 production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PI5-kinase). In addition, c-terminus mutants showed a potentiated regulation by pKa. On the other hand, for the two cytoplasmic-loop mutations, an impaired activation by pKa was observed. The effects of the mutations on pKC stimulation of the channel paralleled the effects on pKa stimulation, suggesting that both regulatory inputs are similarly affected by the mutations. We tested whether PKC-mediated activation of IKs, similarly to the PKA-mediated activation, can regulate the channel response to PIP2. after pKC activation, channel was less sensitive to changes in membrane PIP2 levels, consistent with an increase in apparent channel-PIP2 affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP2 by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP2 production. Our data indicates that stimulation by pKa and pKC can partially rescue LQT1 mutant channels with weakened response to PIP2 by strengthening channel interactions with PIP2.