Role of Astrocytic TREK-2 Potassium Channels in Cerebral Ischemia

描述（申请人提供）：中风是美国第三大死亡原因，每年约有275，000人死亡。缺血性中风导致神经元死亡的主要介质之一是细胞外突触间隙中谷氨酸水平升高，导致兴奋性毒性和神经元细胞死亡。星形胶质细胞是脑中数量最多的细胞，对神经元活力至关重要，并且刺激缺血或缺血后脑中的关键星形胶质细胞功能如谷氨酸和钾稳态可能有助于神经保护。星形胶质细胞中的钾通道主要负责维持星形胶质细胞的超极化膜电位，其允许这些细胞有效地摄取谷氨酸和K+。一种类型的K+通道定位在星形胶质细胞中，可能是相关的在缺血条件下是TREK-2串联孔结构域K+通道。TREK-2通道被多不饱和脂肪酸、生理范围内的细胞内酸中毒和机械拉伸激活。在缺血期间，磷脂酶的活化促进花生四烯酸的释放和积累，星形胶质细胞的细胞内pH变为酸性并且星形胶质细胞肿胀。所有这些变化将激活星形胶质细胞中的TREK-2通道，以帮助在病理事件如缺氧、缺血、缺氧、低血糖和/或扩散性抑郁期间维持细胞外谷氨酸和K+浓度较低。我们的工作假设是TREK-2钾通道在缺血期间的钾缓冲和谷氨酸清除中起主要作用，并且我们的初步数据证明实验性缺血后星形胶质细胞中TREK-2通道的功能上调支持了这一假设。使用多种技术通过RNA干扰、全细胞电压钳、谷氨酸清除试验和体外缺血模型等方法，我们提出直接研究TREK-2通道在正常和病理条件下维持星形胶质细胞膜电位以及缓冲谷氨酸和K+的作用（谷氨酸兴奋毒性、缺氧和低血糖），以及它们在缺血性损伤如中风期间保护神经元的能力。 公共卫生相关性：尽管中风是美国第三大死因，但目前美国约有540万中风幸存者。尽管损伤严重，但许多细胞在最初的几个小时内不会受到不可逆的损伤，并且可以通过早期恢复血流或其他干预措施来挽救。星形胶质细胞是脑中数量最多的细胞，通常执行许多对神经元活力至关重要的功能，并且在缺血或缺血后脑中刺激星形胶质细胞功能如钾缓冲和谷氨酸清除可能潜在地有助于神经保护。所提出的实验的意义在于，它们代表了一种全面的努力，以机械地阐明星形胶质细胞中TREK-2通道在病理生理条件（如中风引起的缺血）期间维持神经元功能中的作用。

DESCRIPTION (provided by applicant): Strokes are the third-leading cause of death in the United States killing about 275,000 people a year. One of the major mediators of neuronal death due to ischemic stroke is the elevated level of glutamate in the extracellular synaptic space which leads to excitotoxicity and neuronal cell death. Astrocytes, the most numerous cells in the brain, are essential for neuronal viability and stimulation of key astrocytic functions such as glutamate and potassium homeostasis in ischemic or post-ischemic brain could potentially contribute to neuroprotection. Potassium channels in astrocytes are predominantly responsible for maintaining the hyperpolarized membrane potential of astrocytes which allows these cells to effectively take up glutamate and K+. One type of K+ channel localized in astrocytes that is likely to be pertinent during ischemic conditions is the TREK-2 tandem-pore domain K+ channel. TREK-2 channels are activated by polyunsaturated fatty acids, intracellular acidosis in the physiological range and by mechanical stretch. During ischemia, activation of phospholipases promotes liberation and accumulation of arachidonic acid, the intracellular pH of astrocytes becomes acidic and astrocytes swell. All of these changes will activate TREK-2 channels in astrocytes to help maintain extracellular glutamate and K+ concentrations low during pathological events such as anoxia, ischemia, hypoxia, hypoglycemia and/or spreading depression. Our working hypothesis is that TREK-2 potassium channels play a major role in potassium buffering and glutamate clearance during ischemia and this hypothesis is supported by our preliminary data demonstrating functional upregulation of TREK-2 channels in astrocytes after experimental ischemia. Using a combination of techniques (such as RNAi, whole cell voltage clamp, glutamate clearance assays and an in vitro model of ischemia), we propose to directly examine the role of TREK-2 channels in maintaining the membrane potential of astrocytes and in buffering glutamate and K+ during normal and pathological conditions (glutamate excitoxicity, anoxia and hypoglycemia), as well as their ability to protect neurons during ischemic insults such as stroke. PUBLIC HEALTH RELEVANCE: Although strokes are the third-leading cause of death in the United States, there are about 5.4 million stroke survivors in the United States today. Despite the severity of the insult, many cells are not irreversibly damaged within the first few hours and can be rescued by early restoration of blood flow or other interventions. Astrocytes, the most numerous cells in the brain, normally perform many functions that are essential for neuronal viability and stimulation of astrocytic functions such as potassium buffering and glutamate clearance in ischemic or post-ischemic brain could potentially contribute to neuroprotection. The significance of the proposed experiments is that they represent a comprehensive effort to mechanistically elucidate the role of TREK-2 channels in astroctyes in the maintenance of neuronal function during pathophysiological conditions such as ischemia due to stroke.