KCNQ Channels and Vasoconstrictor Signal Transduction

描述(申请人提供)：KCNQ K通道与人类疾病有关，从心律失常到先天性耳聋和癫痫。在神经元中，这些通道处于电压敏感的K(Kv)电流之下，该电流被乙酰胆碱负性调节以调节突触后神经元的兴奋性。尽管KCNQ通道以前没有被认为在血管收缩信号转导中发挥作用，但我们最近的研究表明，生理血管收缩药精氨酸加压素(AVP，10-100 pm)对动脉肌细胞兴奋性的调节涉及蛋白激酶C依赖的KCNQ5通道活性的抑制。我们最近还发表了证据表明，这种对KCNQ通道的负性调节是低[AVP](30 PM)在大鼠肠系膜动脉收缩血管作用的基础。以往没有研究探讨KCNQ通道是如何被血管活性激素调节的，涉及的信号转导机制，表达不同通道亚型的血管床其功能或调节是否不同，或者这些通道或信号通路是否可能成为心血管疾病治疗的有用靶点。因此，我们建议：1.鉴定KCNQ家族(Kv7.1-7.5)通道在大鼠肠系膜动脉或基底动脉的表达亚型，并确定它们在调节动脉内径中的功能。实时定量聚合酶链式反应和免疫组织化学方法检测KCNQ通道的表达。通道功能将通过分离动脉的压力肌图和分离的心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.明确AVP(和其他可能的血管收缩激素)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-羟色胺、血管紧张素和苯肾上腺素)对新鲜分离的动脉肌细胞KCNQ电流的时间和浓度依赖效应。我们将利用药物激活剂/抑制剂或分子试剂来研究特定的蛋白激酶C亚型和A激酶锚定蛋白150(AKAP150)在KCNQ电流调节中的作用，以干扰其表达/功能。KCNQ通道上特定残基的磷酸化作用(将通过质谱学鉴定)将通过对培养的平滑肌细胞中的激酶或磷酸化位点进行分子靶向以及在转基因小鼠中敲击调节异常的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标记的KCNQ通道蛋白的免疫共沉淀进行鉴定。3.动脉KCNQ通道在血管收缩和血压调节中起重要作用的假说将通过在体测量选择性KCNQ通道激动剂和阻滞剂对麻醉大鼠或慢性仪器清醒大鼠肠系膜动脉血流和全身血压的影响来验证。

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.