The Role of 2-Pore Domain Potassium Channels in Acute Lung Injury.

描述(申请人提供)：尽管肺病学和重症监护医学取得了进展，但急性肺损伤(ALI)患者的死亡率仍然很高，可能是因为对这种疾病的潜在机制知之甚少。ALI患者通常需要高氧浓度和正压通气，尽管这两种疗法都会加重持续的肺损伤。虽然高氧激活的信号级联是众所周知的，但对肺泡扩张后的“机械转导”却知之甚少。最近的文献表明，2-孔域钾(K2P)通道可能作为机械感受器和机械转导，参与刺激-分泌耦合。然而，对K2P通道在肺中的表达和潜在功能知之甚少。我们提出了一个大胆而新颖的假设，即K2P通道在肺上皮细胞中表达，高氧、机械拉伸和肿瘤坏死因子-α暴露(类似于ALI的环境)引起的致病性K2P通道调节导致上皮细胞炎性介质分泌的失调，以及上皮屏障功能的丧失，这是ALI的两个特征。我们的首要目标是研究导致高氧和机械牵张所致肺损伤的机制，并确定K2P通道作为寻找治疗ALI的创新策略的新靶点。具体地说，我们将采用体外和体内两种方法，包括K2P基因敲除小鼠，[1]利用分子技术、免疫组织化学和膜片钳研究，[1]研究高氧、机械拉伸和肿瘤坏死因子-a对培养的小鼠、大鼠和人的肺泡上皮细胞K2P通道表达和功能的影响，[2]确定K2P通道在培养的和原代肺泡上皮细胞以及K2P基因敲除小鼠的支气管肺泡灌洗液中分泌炎症介质的作用，[3]证明K2P通道通过钙依赖的紧密连接磷酸化来调节上皮屏障功能。我们拥有独特的专业知识和技术能力，可以在体外和体内模型中研究高氧和机械拉伸对ALI的影响。此外，Jaggar博士拥有无与伦比的测量全球和局部细胞内钙浓度的装置。UTHSC的学术环境，杰出的导师，丰富的合作机会，以及机构、部门和部门对我研究成功的承诺，为我提供了智力基础设施和资金支持，以保证我在5年内取得独立研究资金，包括R01奖。这些长期目标将通过以下方式实现：每年至少在国际会议上发表2篇出版物和2篇摘要报告，并辅之以正式的课程作业，以及沃特斯博士、阿南德博士和我的职业咨询委员会的密切指导。

DESCRIPTION (provided by applicant): Despite advances in Pulmonology and Intensive Care Medicine, mortality rates of patients with Acute Lung Injury (ALI) remain high, perhaps because the underlying mechanisms of this disease are poorly understood. Patients with ALI routinely require high oxygen concentrations and positive-pressure ventilation, although both therapies accentuate ongoing lung injury. While the signaling cascades activated by hyperoxia are well known, "mechano-transduction" after alveolar distension is poorly understood. Recent literature suggests that 2-pore domain potassium (K2P) channels may act as mechano-sensors and mechano-transducers, and participate in stimulus-secretion coupling. However, little is known about the expression and potential functions of K2P channels in the lung. We propose a bold and novel hypothesis that K2P channels are expressed in lung epithelial cells, and that pathogenic K2P channel regulation caused by hyperoxia, mechanical stretch and TNF-a exposure, an environment similar to the one encountered in ALI, results in dysregulation of inflammatory mediator secretion from epithelial cells, and in loss of epithelial barrier function, two hallmarks of ALI. Our overarching objective is to investigate the mechanisms leading to hyperoxia- and mechanical stretch- induced lung injury, and to identify K2P channels as a new target in the search for innovative therapeutic strategies against ALI. Specifically, we will use both in vitro and in vivo approaches including K2P knockout mice to [1] investigate the effects of hyperoxia, mechanical stretch and TNF-a on K2P channel expression and function in cultured mouse and primary rat and human alveolar epithelial cells using molecular techniques, immunohistochemistry and patch clamp studies, [2] to determine the role of K2P channels in inflammatory mediator secretion from cultured and primary alveolar epithelial cells, and in broncho-alveolar lavage fluid from K2P knockout mice, and [3] to demonstrate that K2P channels regulate epithelial barrier function via Ca2+-dependent tight junction phosphorylation. We have the unique expertise and technical capabilities to study the effects of hyperoxia and mechanical stretch in both in vitro and in vivo models of ALI. In addition, Dr. Jaggar has an inimitable setup to measure global and localized intracellular Ca2+ concentrations. The academic environment at UTHSC, the outstanding mentorship, rich opportunities for collaborations, and the institutional, departmental, and divisional commitment to my research success provide the intellectual infrastructure and the financial support to guarantee my progress towards independent research funding, including an R01 award, within 5 years. These long-term goals will be achieved by targeting a minimum of 2 publications and 2 abstract presentations per year at international meetings, supplemented by formal coursework, and the close mentorship of Dr. Waters, Dr. Anand and my Career Advisory Committee.