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因，每年导致约275,000人死亡。细胞外突触间隙谷氨酸水平升高是缺血性卒中神经元死亡的主要介质之一，导致兴奋性毒性和神经细胞死亡。星形胶质细胞是脑中数量最多的细胞，对神经元的存活至关重要，在脑缺血或缺血后刺激关键的星形胶质细胞功能，如谷氨酸和钾的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，使这些细胞有效地摄取谷氨酸和K+。在星形胶质细胞中定位的一种K+通道可能在缺血条件下是相关的，那就是Trek-2串联孔域K+通道。TREK-2通道被多不饱和脂肪酸、生理范围内的细胞酸中毒和机械拉伸所激活。在缺血时，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变酸性，星形胶质细胞肿胀。所有这些变化都将激活星形胶质细胞上的Trek-2通道，以帮助在缺氧、缺血、缺氧、低血糖和/或扩散性抑郁等病理事件中保持细胞外谷氨酸和K+的低浓度。我们的工作假设是，在缺血期间，Trek-2钾通道在钾缓冲和谷氨酸清除中起主要作用，这一假设得到了我们的初步数据的支持，该数据表明实验性缺血后星形胶质细胞中Trek-2通道的功能上调。结合多种技术(如RNAi、全细胞电压钳、谷氨酸清除试验和体外缺血模型)，我们建议直接研究Trek-2通道在正常和病理条件下(谷氨酸兴奋性、缺氧和低血糖)在维持星形胶质细胞膜电位、缓冲谷氨酸和K+方面的作用，以及它们在脑缺血损伤(如中风)中保护神经元的能力。 与公共卫生相关：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。尽管侮辱的严重性，许多细胞在最初的几个小时内并不是不可逆转的受损，可以通过早期恢复血流或其他干预措施来挽救。星形胶质细胞是大脑中数量最多的细胞，通常具有许多对神经元生存至关重要的功能，而刺激星形胶质细胞的功能，如脑缺血或缺血后的钾缓冲和谷氨酸清除，可能有助于神经保护。提出的实验的意义在于，它们代表了一项全面的努力，以机械地阐明星形胶质细胞中Trek-2通道在中风引起的缺血等病理生理条件下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.