KCNQ Channels in Airway Smooth Muscle Physiology and Disease

 描述(由申请人提供) 哮喘是一种慢性呼吸系统疾病，以可逆性呼吸道阻塞和对支气管收缩刺激的高反应性为特征。退伍军人代表了一大批哮喘患者，其中许多人无法通过现有的治疗方法缓解症状。哮喘的可逆性气道阻塞主要是由于肺细支气管处的气道平滑肌细胞(ASMC)过度收缩所致。ASMC过度收缩的机制尚不清楚，但可能涉及对GQ/11偶联受体激动剂的过度支气管收缩反应，这些受体激动剂包括乙酰胆碱、组胺、内皮素-1和白三烯D4。通过电压敏感的钙通道(VSCCs)增加的钙内流导致了过度的支气管收缩反应。激活VSCCs所需的电压变化可以通过抑制钾(K+)通道活动来提供。我们最近发现，KCNQ(KV7家族)K+通道在新鲜分离的细支气管肌细胞中表达和激活，并且其活性被多种支气管收缩激动剂抑制。我们进一步证明，KV7电流抑制足以在精密切割的人肺切片上诱导气道收缩，并且药物KV7通道激动剂显著减轻气道收缩。基于这些发现，我们建议验证一种假设，即支气管收缩激动剂抑制ASMC中KV7家族K+通道的活动，从而诱导膜去极化和ASMC过度收缩。此外，我们将探索另外两个假设，这两个假设对于我们对哮喘的理解和治疗具有重要的临床/翻译相关性。我们假设炎症介质改变了KV7通道的表达，这在哮喘的气道高反应性的病因学中发挥了作用。此外，由于KV7通道是已确定的药物靶点，我们假设临床上使用的KV7通道激活剂可以作为单一疗法使用，或者它们可以与其他疗法联合使用，以防止或减少哮喘患者过度的气道收缩。为了解决我们的假设，我们提出了三个具体目标。目的1)阐明GQ/11偶联受体激动剂调节ASMCs KV7电流的机制。蛋白激酶C的作用将通过使用各种生化、药理学和分子生物学方法，结合膜片钳电生理学和对人和豚鼠ASMCs和肺组织的气道平滑肌功能的测量来确定。目的2)验证两种新的假设，将气道炎症与抑制ASMCs中KV7通道的表达和增加对支气管收缩刺激的敏感性联系起来。假设1：炎症介质，如IL-13和IL-1β，上调抑制因子1沉默转录因子(REST)，从而抑制ASMC中KCNQ基因的转录。假设2：微小RNA 146a(MiR146a)，也是炎症介质的反应增加，诱导转录后抑制Kv7.5在ASMC的表达。REST和miR146a的表达将在正常人和哮喘患者和豚鼠肺的ASMC中进行测量/改变，以确定它们与KV7通道表达/功能改变的关系以及与呼吸道高反应性的联系。目的3)评价KV7通道激动剂单独或与β-2受体激动剂合用对正常和抗原致敏豚鼠体内气道功能的影响。我们提出的研究具有重要意义，因为它们将阐明以前未知的ASMC过度收缩的机制，并确定一种新的治疗策略，以更好地缓解哮喘患者的气道高反应性。这一结果将直接惠及数以千计患有哮喘或其他呼吸道疾病的退伍军人和平民。

 DESCRIPTION (provided by applicant) Asthma is a chronic respiratory disease characterized by reversible airway obstruction and hyper- responsiveness of the airways to bronchoconstrictor stimuli. Veterans represent a large cohort of asthma sufferers, many of whom do not achieve relief of symptoms with available therapies. Reversible airway obstruction in asthma results primarily from hypercontraction of airway smooth muscle cells (ASMCs) in the bronchioles of the lung. The mechanisms involved in ASMC hypercontraction are poorly characterized but likely involve exaggerated bronchoconstrictor responses to Gq/11-coupled receptor agonists, which include acetylcholine, histamine, endothelin-1, and leukotriene D4. Increased Ca2+ influx via voltage-sensitive Ca2+ channels (VSCCs) contributes to the exaggerated bronchoconstrictor responses. The voltage change required to activate VSCCs can be provided by suppressing potassium (K+) channel activity. We recently discovered that KCNQ (Kv7 family) K+ channels are expressed and active in freshly isolated bronchiolar myocytes and that their activity is suppressed by multiple bronchoconstrictor agonists. We further demonstrated that Kv7 current suppression is sufficient to induce airway constriction and that pharmacological Kv7 channel activators significantly attenuate airway constriction in precision-cut human lung slices. Based on these discoveries, we propose to examine the hypothesis that bronchoconstrictor agonists suppress the activity of Kv7 family K+ channels in ASMCs to induce membrane depolarization and ASMC hypercontraction. Furthermore, we will explore two additional hypotheses that have important clinical/translational relevance for our understanding and treatment of asthma. We hypothesize that inflammatory mediators alter Kv7 channel expression, which plays a role in the etiology of airway hyperresponsiveness in asthma. Furthermore, because Kv7 channels are established drug targets, we hypothesize that clinically used Kv7 channel activators can be used as a monotherapy or they may be combined with other therapies to prevent or reduce excessive airway constriction in asthma. To address our hypotheses, three Specific Aims are proposed. Aim 1) Elucidate the mechanisms by which Gq/11-coupled receptor agonists regulate Kv7 currents in ASMCs. The role of protein kinase C will be determined by using a variety of biochemical, pharmacological, and molecular biological approaches in combination with patch clamp electrophysiology and measurements of airway smooth muscle function in human and guinea pig ASMCs and lung tissues. Aim 2) Test two novel hypotheses linking airway inflammation to suppression of Kv7 channel expression in ASMCs and increased sensitivity to bronchoconstrictor stimuli. Hypothesis 1: inflammatory mediators, such as IL-13 and IL-1β, upregulate repressor element 1 silencing transcription factor (REST), which then inhibits transcription of KCNQ genes in ASMCs. Hypothesis 2: micro RNA 146a (miR146a), which is also increased in response to inflammatory mediators, induces post- transcriptional repression of Kv7.5 expression in ASMCs. REST and miR146a expression will be measured/altered in ASMCs from normal and asthmatic human and guinea pig lungs to determine their relationship to altered expression/function of Kv7 channels and the link to airway hyperresponsiveness. Aim 3) Evaluate the therapeutic benefits of Kv7 channel activators, alone or combined with β2-adrenergic receptor agonists, on airway function in vivo in normal and antigen-sensitized guinea pigs. Our proposed studies are significant because they will elucidate previously unknown mechanisms for ASMC hypercontraction and identify a new therapeutic strategy to provide better relief of airway hyperresponsiveness in asthma patients. This outcome would directly benefit thousands of veterans and civilians who suffer from asthma or other airway diseases.