Acid-sensing ion channels and ischemic brain injury

描述(由申请人提供)：脑缺血的特征是显著的、迅速的pH下降，尽管通过多种或不确定的机制，这被认为是有害的。近年来在脑内发现的酸性感觉离子通道(ASICs)在突触神经传递中具有生理功能，它提供了一个弥漫的、基于膜的、受体门控的离子通道系统，它将对脑缺血时的病理性pH下降做出反应。这些H受体门控通道是钠通道，其中一部分也是钙离子通透性的。酸敏感性和钙通透性提示在缺血性脑损伤中起一定作用。使用膜片钳技术和大脑皮层培养的天然神经元的钙成像，我们显示了这些通道的pH敏感性和酸诱导的钙摄取。在模拟缺血(氧糖剥夺或NaCN)的背景下，酸诱导的通道电流和钙摄取均显著增强。因此，这些通道以一种相互增强的方式对酸中毒和“缺血”作出反应。通道电流和钙摄取不依赖谷氨酸，可被ASIC药物阻断和ASIC 1a通道亚单位特异性阻断所抑制。因此，我们的初步研究支持新的细胞和分子机制介导的缺血-酸中毒所致的脑损伤，我们提供了以下特定目的的解剖：单细胞记录和单个ASIC亚单位cDNAs的转导将显示：1)钙通透性ASICs通过亚单位特异性机制在模拟缺血中产生细胞损伤。利用大鼠和小鼠全脑缺血模型以及ASIC1a和ASIC2a基因敲除小鼠，我们提出：2)阻断ASICs对在体缺血性脑损伤有保护作用。利用包括定点突变在内的分子技术，我们将描述内源性锌对钙通透性ASICs的调控：3)酸中毒和缺血诱导的细胞损伤中高亲和力锌对ASICs电流的调控。这些实验将描述不依赖谷氨酸的新的缺血性脑损伤的机制，以及缺血性酸中毒的中心作用。ASIC阻断将为中风提供新的、有效的潜在治疗方法。

DESCRIPTION (provided by applicant): Brain ischemia is characterized by a marked, rapid fall in pH, which is assumed to be injurious although through multiple or uncertain mechanisms. The recent discovery in brain of Acid Sensing Ion Channels (ASICs), which are ubiquitous and function physiologically in synaptic neurotransmission, offers a diffuse, membrane based, receptor gated ion channel system, which will respond to the pathologic pH fall in brain ischemia. These H+ receptor gated channels are Na+ channels, a portion of which is also Ca2+ permeable. Acid sensitivity and Ca2+ permeability suggest a role in ischemic brain injury. Using patch clamp techniques and Ca2+ imaging of native neurons in cortical cultures we show the pH sensitivity of these channels and acid induced Ca2+ uptake. Both the acid induced channel currents and Ca2+ uptake are greatly potentiated in the setting of modeled ischemia (Oxygen Glucose Deprivation-OGD or NaCN). Thus these channels respond to both acidosis and "ischemia" in a mutually potentiating manner. The channel current and Ca2+ uptake are glutamate independent, are inhibited by ASIC pharmacologic blockade and by specific blockade of the ASIC 1a channel subunit. Thus, our preliminary studies support new cellular and molecular mechanisms mediating ischemic-acidosis induced brain injury, which we offer to dissect with the following Specific Aims: Single cell recording and transfection of individual ASIC subunit cDNAs will show that: 1) Ca2+ permeable ASICs produce cell injury in modeled ischemia via a subunit specific mechanism. Using rat and mouse global ischemia models and ASIC1a & ASIC2a knockout mice, we propose that: 2) Blockade of ASICs protects against ischemic brain injury in vivo. With molecular techniques including site directed mutagenesis we will describe endogenous Zn2+ modulation of Ca2+permeable ASICs: 3) Characterize high-affinity Zn2+ modulation of ASIC currents in acidosis and ischemia -induced cell injury. These experiments will describe new, glutamate independent, mechanisms of ischemic brain injury and the central role of ischemic-acidosis. ASIC blockade will offer new and potent potential therapy for stroke.