Role of Astrocytic TREK-2 Potassium Channels in Cerebral Ischemia

描述（由申请人提供）：中风是美国第三大死因，每年约有27.5万人死于中风。细胞外突触间隙谷氨酸水平升高是缺血性脑卒中神经元死亡的主要介质之一，其导致兴奋毒性和神经元细胞死亡。星形胶质细胞是大脑中数量最多的细胞，对神经元的活力至关重要，刺激星形胶质细胞的关键功能，如缺血或缺血后大脑的谷氨酸和钾稳态，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，使这些细胞有效地吸收谷氨酸和K+。星形胶质细胞中的一种K+通道是TREK-2串联孔结构域K+通道，可能与缺血条件有关。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。缺血时，磷脂酶的激活促进花生四烯酸的释放和积累，星形胶质细胞内pH变酸，星形胶质细胞膨胀。所有这些变化将激活星形胶质细胞中的TREK-2通道，以帮助在缺氧、缺血、缺氧、低血糖和/或播散性抑郁等病理事件中维持细胞外谷氨酸和K+的低浓度。我们的工作假设是TREK-2钾通道在缺血时钾缓冲和谷氨酸清除中起主要作用，我们的初步数据显示实验性缺血后星形胶质细胞中TREK-2通道功能上调，这一假设得到了支持。利用RNAi、全细胞电压钳、谷氨酸清除试验和体外缺血模型等综合技术，我们建议直接研究TREK-2通道在正常和病理状态（谷氨酸兴奋性、缺氧和低血糖）下维持星形胶质细胞膜电位和缓冲谷氨酸和K+的作用，以及它们在缺血性损伤（如中风）期间保护神经元的能力。

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.