KCNQ Channels and Vasoconstrictor Signal Transduction

描述（由申请人提供）：KCNQ K+通道涉及从心律失常到先天性耳聋和癫痫等人类疾病。在神经元中，这些通道是电压敏感的K+ (Kv)电流的基础，该电流由乙酰胆碱负调节，以调节突触后神经元的兴奋性。虽然KCNQ通道以前没有被认为在血管收缩信号转导中起作用，但我们最近的研究表明，精氨酸血管加压素（AVP, 10-100 pM）的生理血管收缩浓度对动脉肌细胞兴奋性的调节涉及对KCNQ5通道活性的蛋白激酶c依赖性抑制。我们最近也发表了证据表明，KCNQ通道的负调控是大鼠肠系膜动脉低AVP （30pm）血管收缩作用的基础。以前没有研究调查过血管活性激素如何调节KCNQ通道，所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中存在差异，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动脉肌细胞中表达的KCNQ家族（Kv7.1- 7.5）通道亚型，并确定其在调节动脉直径中的功能作用。实时PCR和免疫组化检测KCNQ通道的表达。通道功能将通过孤立动脉的压力肌图和孤立肌细胞的膜片钳电生理来评估。选择性KCNQ通道阻滞剂和激活剂以及分子敲除方法将用于评估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）对新分离动脉肌细胞中KCNQ电流的时间和浓度依赖性作用。特异性蛋白激酶C异构体和A激酶锚定蛋白150 （AKAP150）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。KCNQ通道上特定残基磷酸化的作用（将通过质谱鉴定）将通过分子靶向培养平滑肌细胞中的激酶或磷酸化位点以及敲除转基因小鼠中失调的KCNQ通道来评估。信号复合体中与KCNQ通道相关的分子将通过与天然或flag标记的KCNQ通道蛋白共免疫沉淀来鉴定。3. 最后，动脉KCNQ通道在血管收缩作用和血压调节中发挥重要作用的假设将通过测量选择性KCNQ通道激活剂和阻滞剂对急性麻醉大鼠或长期麻醉清醒大鼠肠系膜动脉血流和全身血压的影响来验证。公共卫生相关性：KCNQ通道主要因其在神经元兴奋中的作用而得到认可。KCNQ通道的激活剂或阻滞剂已分别用于癫痫和阿尔茨海默病的临床治疗。这些药物对动脉收缩的影响及其作为血管收缩激素作用介质的作用（在我们的初步结果中首次被证明）尚未得到重视，可能对KCNQ通道调节剂在现有治疗中的使用以及它们在心血管疾病治疗中的潜在应用具有重要意义。

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. PUBLIC HEALTH RELEVANCE: KCNQ channels have been recognized primarily for their role in neuronal excitation. Activators or blockers of KCNQ channels have been used clinically for treatment of epilepsy and Alzheimer's disease, respectively. The effects of these drugs on arterial constriction and their role as mediators of vasoconstrictor hormone action (demonstrated for the first time in our preliminary results) have not been appreciated and might have important implications for the use of KCNQ channel modulators in existing therapies as well as for their potential use in the treatment of cardiovascular diseases.