KCNQ Channels and Vasoconstrictor Signal Transduction

KCNQ 通道和血管收缩信号转导

基本信息

批准号：
8206590
负责人：
KENNETH L BYRON
金额：
$ 37.94万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-15 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8206590
关键词：
A kinase anchoring protein Acetylcholine Action Potentials Address Adrenergic Agonists Affect Agonist Alzheimer&apos s Disease Angiotensin II Argipressin Arrhythmia Arteries Binding Biochemical Blood Pressure Blood Vessels Blood flow Brain Caliber Cardiac Cardiac Myocytes Cardiovascular Diseases Carotid Arteries Cell Line Characteristics Co-Immunoprecipitations Complex Conscious Down-Regulation Electrocardiogram Electrophysiology (science)Environment Epilepsy Family Family member Generations Genes Hormones Immunohistochemistry In Vitro Individual Knock-in Mouse Labyrinth Long QT Syndrome Lung Maleimides Mass Spectrum Analysis Measures Mediator of activation protein Membrane Membrane Potentials Mesenteric Arteries Mesentery Methods Microspheres Mink Modeling Molecular Molecular Target Monitor Mus Muscarinic Acetylcholine Receptor Muscle Cells Mutation Myocardium Myography Names Neurons Neurotransmitters Perfusion Pharmaceutical Preparations Phenylephrine Phosphorylation Phosphorylation Site Phosphotransferases Physiological Play Potassium Channel Process Protein Isoforms Protein Kinase C Proteins Publishing RNA Interference Rattus Reagent Regulation Rest Role Serotonin Serotonin Agonists Signal Pathway Signal Transduction Signal Transduction Pathway Skeletal Muscle Smooth Muscle Smooth Muscle Myocytes Structure Sympathetic Ganglia System Techniques Testing Thoracic aorta Time Tissues Transgenic Mice Vasoconstrictor Agents Visceral Voltage-Gated Potassium Channel basilar artery blood pressure regulation cardiovascular disorder therapy channel blockers congenital deafness constriction femoral artery flupirtine human disease in vivo inhibitor/antagonist instrument intravital microscopy knock-down linopirdine loss of function member mouse model neuronal excitability neurotransmitter release novel patch clamp postsynaptic pressure protein expression public health relevance response vascular bed vasoconstriction voltage

项目摘要

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.

描述（由申请人提供）：KCNQ K+通道与人类疾病有关，从心律不齐到先天性耳聋和癫痫病。在神经元中，这些通道是对电压敏感的K+（KV）电流的基础，该电流受乙酰胆碱对调节突触后神经元兴奋性的负调节。尽管KCNQ通道以前尚未被认为在血管收缩信号转导中发挥作用，但我们最近表明，通过生理血管促成浓度的精氨酸加压素（AVP，AVP，10-100 PM）调节动脉肌细胞的兴奋性涉及蛋白质 - 基因酶C依赖于KCCNQ5通道的抑制。我们最近还发表了证据，表明，这种KCNQ通道的阴性调节是大鼠肠系膜动脉中低[AVP]（30 pm）的血管收缩作用的基础。先前的研究尚未检查如何通过血管活性激素，涉及的信号转导机制来调节KCNQ通道，它们的功能还是调节在表达不同通道亚型的血管床之间是否有所不同，或者这些通道或信号传导途径是否可能是对心血管疾病治疗的有用靶标。因此，我们建议：1。鉴定在大鼠肠系膜或基底动脉的动脉肌细胞中表达的KCNQ家族的亚型（KV7.1-7.5）通道的亚型，并确定其在调节动脉直径的功能作用。实时PCR和免疫组织化学将用于检测KCNQ通道表达。通道功能将通过孤立的动脉压力密闭和斑块电生理学的压力肌照相评估。选择性KCNQ通道阻滞剂和激活剂以及分子敲低方法将用于评估特定通道亚型的功能。 2。确定AVP（以及可能其他血管收缩激素）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中KCNQ电流的时间和浓度依赖性作用。特异性蛋白激酶C同工型和激酶锚定蛋白150（AKAP150）在KCNQ电流调节中的作用将使用药理学激活剂/抑制剂或分子试剂进行研究以破坏其表达/功能。特异性残基在KCNQ通道上的磷酸化作用（通过质谱法鉴定）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的分子靶向，并通过敲击转基因小鼠中的KCNQ通道的失调KCNQ通道。与KCNQ通道相关的分子，将使用与天然或标记的KCNQ通道蛋白的共免疫沉淀来鉴定。 3.最后，将通过测量选择性KCNQ通道激活剂和阻滞剂对肠系膜动脉血流的体内影响和阻滞剂在麻醉大鼠或在切入有意识的仪器中急性测量的肠系膜动脉血流和全身性血压的体内影响来测试的假设在血管收缩的作用和血压调节中起重要作用。公共卫生相关性：KCNQ渠道主要因其在神经元激发中的作用而被认可。 KCNQ通道的激活剂或阻滞剂分别用于治疗癫痫病和阿尔茨海默氏病。这些药物对动脉收缩的影响及其作为血管收缩激素作用的介体（在我们的初步结果中首次证明）的作用尚未得到欣赏，并且可能对使用KCNQ通道调节剂在现有疗法中的使用以及其在治疗心血管疾病的潜在使用中具有重要意义。