Cardiovascular Research Institute, Department of Medicine, University of Rochester, Rochester, NY, USA.
Channels (Austin). 2010 Jan-Feb;4(1):3-11. doi: 10.4161/chan.4.1.10227. Epub 2010 Jan 5.
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 (PIP(2)). These regulatory pathways were shown to crosstalk, with PKA phosphorylation increasing the apparent affinity of IKs to PIP(2). Here we study the effects of LQT1 mutations in putative PIP(2)-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 PIP(2) levels, consistent with a decrease in apparent channel-PIP(2) affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP(2)-depletion and more sensitive to stimulation of PIP(2) 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 PIP(2). After PKC activation, channel was less sensitive to changes in membrane PIP(2) levels, consistent with an increase in apparent channel-PIP(2) affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP(2) by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP(2) production. Our data indicates that stimulation by PKA and PKC can partially rescue LQT1 mutant channels with weakened response to PIP(2) by strengthening channel interactions with PIP(2).
长 QT 综合征导致扭转型室性心动过速、心室颤动和猝死。长 QT 综合征最常见的遗传形式 LQT1 是由于钾通道基因 KCNQ1 的突变引起的，该基因形成了主要的复极化心脏 K(+)通道之一，即 IKs。已经表明 IKs 受到β肾上腺素能受体的调节，通过蛋白激酶 A（PKA），以及通过 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（PIP(2)）。这些调节途径被证明存在串扰，PKA 磷酸化增加了 IKs 对 PIP(2)的表观亲和力。在这里，我们研究了 LQT1 突变在假定的 PIP(2)-KCNQ1 相互作用位点对 PKA 和 GqPCR 对 IKs 调节的影响。对于位于 KCNQ1 亚基四个不同细胞质域中的保守、带正电荷的氨基酸中的 LQT1 突变，测试了它们对 IKs 调节的影响：R174C（S2-S3）、R243C（S4-S5）、R366Q（近端 C 末端）和 R555C（远端 C 末端）。IKs （近端和远端）C 末端的突变增加了通道对膜 PIP(2)水平变化的敏感性，这与通道-PIP(2)亲和力的表观降低一致。这些突变通道对受体介导的 PIP(2)耗竭引起的抑制更敏感，对磷脂酰肌醇-4-磷酸-5-激酶（PI5-kinase）过表达引起的 PIP(2)产生的刺激更敏感。此外，C 末端突变体显示出 PKA 调节的增强。另一方面，对于两个细胞质环突变，观察到 PKA 激活的受损。突变对 PKC 刺激通道的影响与对 PKA 刺激的影响相似，表明这两种调节输入都受到突变的类似影响。我们测试了 PKC 介导的 IKs 激活是否可以类似于 PKA 介导的激活来调节通道对 PIP(2)的反应。PKC 激活后，通道对膜 PIP(2)水平变化的敏感性降低，与通道-PIP(2)亲和力的表观增加一致。PKC 激活的通道对由脂质激酶抑制剂wortmannin 阻断 PIP(2)合成引起的抑制更不敏感，对 PIP(2)产生的刺激也不敏感。我们的数据表明，PKA 和 PKC 的刺激可以通过加强通道与 PIP(2)的相互作用，部分挽救对 PIP(2)反应减弱的 LQT1 突变体通道。
