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：
7582000
负责人：
KENNETH L BYRON
金额：
$ 39.1万
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/7582000
关键词：
A kinase anchoring protein Acetylcholine Action Potentials Address Adrenergic Agonists Affect 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 Research 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的 K+ (Kv) 电流的基础，该电流受到乙酰胆碱的负调节，以调节突触后神经元的兴奋性。虽然 KCNQ 通道以前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，精氨酸加压素（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蛋白激酶 C- KCNQ5 通道活性的依赖性抑制。我们最近还发表了证据，表明 KCNQ 通道的这种负调节是大鼠肠系膜动脉中低 [AVP] (30 pM) 血管收缩作用的基础。之前没有研究探讨 KCNQ 通道如何受血管活性激素调节、涉及的信号转导机制、表达不同通道亚型的血管床之间它们的功能或调节是否不同，或者这些通道或信号通路是否可能是心血管疾病的有用靶点疗法。因此，我们建议： 1. 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，并确定其在调节动脉直径中的功能作用。实时PCR和免疫组织化学将用于检测KCNQ通道表达。将通过离体动脉的压力肌电描记法和离体肌细胞的膜片钳电生理学来评估通道功能。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估特定通道亚型的功能。 2. 确定 AVP（以及可能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 和去氧肾上腺素）对新鲜分离的动脉肌细胞中 KCNQ 电流的时间和浓度依赖性影响。将使用药理学激活剂/抑制剂或分子试剂破坏其表达/功能来研究特定蛋白激酶 C 亚型和 A 激酶锚定蛋白 150 (AKAP150) 在 KCNQ 电流调节中的作用。 KCNQ 通道上特定残基磷酸化的作用（通过质谱法鉴定）将通过培养平滑肌细胞中激酶或磷酸化位点的分子靶向以及通过敲入转基因小鼠中失调的 KCNQ 通道来评估。信号复合物中与 KCNQ 通道相关的分子将通过与天然或 FLAG 标记的 KCNQ 通道蛋白的免疫共沉淀来鉴定。 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 通道激活剂和阻断剂对麻醉大鼠急性测量的肠系膜动脉血流和全身血压的体内影响来检验或在长期接受仪器检测的有意识的老鼠中。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通道的激活剂或阻断剂在临床上已分别用于治疗癫痫和阿尔茨海默病。这些药物对动脉收缩的作用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得到证实）尚未得到重视，并且可能对 KCNQ 通道调节剂在现有疗法中的使用以及因其在治疗心血管疾病方面的潜在用途。