KCNQ Channels and Vasoconstrictor Signal Transduction

描述（由申请人提供）：KCNQ K+通道与心律失常、先天性耳聋和癫痫等人类疾病有关。在神经元中，这些通道是电压敏感性K+（Kv）电流的基础，其由乙酰胆碱负调节以调节突触后神经元兴奋性。虽然KCNQ通道以前没有被认为在血管收缩信号转导中发挥作用，但我们最近表明，生理性血管收缩剂浓度的精氨酸加压素（AVP，10-100 pM）对动脉肌细胞兴奋性的调节涉及蛋白激酶C依赖性的KCNQ 5通道活性抑制。我们最近也发表了证据表明，KCNQ通道的这种负调节是低[AVP]（30 pM）在大鼠肠系膜动脉中的血管收缩作用的基础。以前的研究还没有检查KCNQ通道如何受血管活性激素的调节，涉及的信号转导机制，它们的功能或调节是否在表达不同通道亚型的血管床之间存在差异，或者这些通道或信号通路是否可能是心血管疾病治疗的有用靶点。因此，我们建议：鉴定大鼠肠系膜或基底动脉的动脉肌细胞中表达的KCNQ家族（Kv7.1- 7.5）通道的亚型，并确定它们在调节动脉直径中的功能作用。将使用真实的时间PCR和免疫组织化学来检测KCNQ通道表达。将通过压力肌描记术在分离的动脉中和膜片钳电生理学在分离的肌细胞中评估通道功能。选择性KCNQ通道阻断剂和激活剂以及分子敲低方法将用于评价特定通道亚型的功能。2.确定AVP（和潜在的其他血管收缩激素）调节KCNQ通道的信号转导机制。我们将测量AVP和其他血管收缩激动剂（5-HT，AngII和苯肾上腺素）对新鲜分离的动脉肌细胞KCNQ电流的时间和浓度依赖性作用。特定的蛋白激酶C亚型和A激酶锚定蛋白150（AKAP 150）在KCNQ电流调节中的作用将使用药理学激活剂/抑制剂或分子试剂来破坏它们的表达/功能。将通过分子靶向培养的平滑肌细胞中的激酶或磷酸化位点以及通过敲除转基因小鼠中失调的KCNQ通道来评估KCNQ通道上特定残基的磷酸化作用（通过质谱法鉴定）。与信号复合物中的KCNQ通道相关的分子将使用与天然或FLAG标记的KCNQ通道蛋白的共免疫沉淀来鉴定。3.最后，动脉KCNQ通道在血管收缩作用和血压调节中起重要作用的假设将通过测量选择性KCNQ通道激活剂和阻断剂对麻醉大鼠或慢性仪器清醒大鼠急性肠系膜动脉血流量和全身血压的体内影响进行测试。

DESCRIPTION (provided by applicant): KCNQ K+ channels have been implicated in human diseases ranging from cardiac arrhythmias to congenital deafness and epilepsy. In neurons, these channels underlie a voltage-sensitive K+ (Kv) current, which is negatively regulated by acetylcholine to regulate postsynaptic neuronal excitability. Although KCNQ channels had not previously been considered to play a role in vasoconstrictor signal transduction, we have recently shown that suggest that regulation of arterial myocyte excitability by physiological vasoconstrictor concentrations of arginine vasopressin (AVP, 10-100 pM) involves protein-kinase C-dependent suppression of KCNQ5 channel activity. We have also recently published evidence that this negative regulation of KCNQ channels underlies the vasoconstrictor actions of low [AVP] (30 pM) in rat mesenteric arteries. No previous studies have examined how KCNQ channels may be regulated by vasoactive hormones, the signal transduction mechanisms involved, whether their functions or regulation differ among vascular beds that express different channel subtypes, or whether these channels or signaling pathways may be useful targets for cardiovascular disease therapies. We therefore propose to: 1. Identify the subtypes of KCNQ family (Kv7.1- 7.5) channels expressed in arterial myocytes from rat mesenteric or basilar arteries and determine their functional roles in regulating artery diameter. Real time PCR and immunohistochemistry will be used to detect KCNQ channel expression. Channel function will be assessed by pressure myography in isolated arteries and patch clamp electrophysiology in isolated myocytes. Selective KCNQ channel blockers and activators and molecular knock-down approaches will be used to evaluate function of specific channel subtypes. 2. Identify the signal transduction mechanisms by which AVP (and potentially other vasoconstrictor hormones) regulate KCNQ channels. We will measure time- and concentration-dependent effects of AVP and other vasoconstrictor agonists (5-HT, AngII, and phenylephrine) on KCNQ currents in freshly isolated arterial myocytes. The roles of specific protein kinase C isoforms and A kinase-anchoring protein 150 (AKAP150) in KCNQ current regulation will be investigated using pharmacological activators/inhibitors or molecular reagents to disrupt their expression/function. The role of phosphorylation of specific residues on KCNQ channels (to be identified by mass spectrometry) will be evaluated by molecular targeting of the kinase or phosphorylation sites in cultured smooth muscle cells and by knocking in dysregulated KCNQ channels in transgenic mice. Molecules associated with KCNQ channels in signaling complexes will be identified using co-immunoprecipitation with native or FLAG-tagged KCNQ channel proteins. 3. Finally, the hypothesis that arterial KCNQ channels play an important role in vasoconstrictor actions and blood pressure regulation will be tested by measuring in vivo effects of selective KCNQ channel activators and blockers on mesenteric artery blood flow and systemic blood pressure measured acutely in anesthetized rats or in chronically instrumented conscious rats.