Acid-sensing ion channels and ischemic brain injury

描述（由申请人提供）：脑缺血的特点是pH值显著、快速下降，尽管通过多种或不确定的机制，但被认为是有害的。最近在脑内发现的酸感离子通道（Acid Sensing Ion Channels, asic）在突触神经传递中普遍存在并发挥着生理功能，它提供了一个弥漫性的、基于膜的、受体门控的离子通道系统，可以对脑缺血时病理性pH下降做出反应。这些H+受体门控通道是Na+通道，其中一部分也是Ca2+可渗透的。酸敏感性和Ca2+通透性提示其在缺血性脑损伤中的作用。使用膜片钳技术和钙离子成像的原生神经元在皮质培养中，我们显示这些通道的pH敏感性和酸诱导Ca2+摄取。在模拟缺血（氧葡萄糖剥夺- ogd或NaCN）的情况下，酸诱导的通道电流和Ca2+摄取都大大增强。因此，这些通道以相互增强的方式对酸中毒和“缺血”作出反应。通道电流和Ca2+摄取是谷氨酸独立的，被ASIC药物阻断和ASIC 1a通道亚单位的特异性阻断所抑制。因此，我们的初步研究支持了介导缺血-酸中毒诱导脑损伤的新的细胞和分子机制，我们提出了以下具体目的：单细胞记录和单个ASIC亚基cdna的转染将表明：1)Ca2+渗透性ASIC通过亚基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敲除小鼠，我们提出：2)阻断asic对体内缺血性脑损伤具有保护作用。通过包括位点定向诱变在内的分子技术，我们将描述内源性Zn2+对Ca2+可渗透性ASIC的调节：3)在酸中毒和缺血诱导的细胞损伤中描述高亲和力Zn2+对ASIC电流的调节。这些实验将描述新的、不依赖谷氨酸的缺血性脑损伤机制和缺血性酸中毒的核心作用。ASIC阻断将为脑卒中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.