Ion channel role-glutamate-independent neuronal injury

描述(申请人提供)：中风和全脑缺血是发病率和死亡率的主要原因。缺血会导致神经元的进行性死亡，即使在再灌流后也是如此。这种迟发性神经元死亡在很大程度上是由于过量的钙进入神经元。多年来，治疗策略一直以钙离子通透性谷氨酸受体门控通道为主要靶点。然而，通过治疗使用谷氨酸受体拮抗剂来防止细胞死亡的努力一直令人失望。虽然谷氨酸受体拮抗剂的耐受性和时机问题在很大程度上导致了临床试验的失败，但一个新的新概念也支持这样的观点，即其他来源的钙离子进入可能在决定缺血性神经元死亡方面同样重要。我们现在有强有力的证据表明，缺血时产生的氧化应激和氧自由基激活了皮层神经元上一种新的钙离子通透性阳离子通道(自由基激活通道：FRAC)，我们的初步数据表明，该通道可能是导致缺血性神经元死亡的谷氨酸非依赖性延迟钙超载的原因。我们的目标是充分描述这一通道的离子特性、药理学特征和调节。我们将尝试确定负责通道激活的特定形式的自由基以及自由基如何激活FRAC的机制。同时使用钙离子成像和膜片钳记录来量化通过Fras的钙离子内流。我们还将确定自由基如何改变急性！分离的神经元和海马片中的神经元。最后，使用体外和最终体内的缺血模型，我们将确定阻止FRAS的激活是否可以保护神经元免受缺血性死亡的影响。一般假设：氧自由基激活钙离子通透性阳离子通道(FRAC)至少部分地导致了缺血性神经元死亡中的谷氨酸非依赖性、延迟性钙超载。骨折的电生理特性。(2)FRA Cs的药理特性和细胞内调节。FRAC激活诱导的钙反应的荧光成像研究。(4)FRAC激活在谷氨酸非依赖性缺血性神经元损伤中的潜在作用我们的长期目标是识别除谷氨酸受体之外的新靶点，以及保护脑细胞免受中风损害的新策略。

DESCRIPTION (provided by applicant): Stroke and global brain ischemia are leading causes of morbidity and mortality. Ischemia causes progressive neuronal death, even after re-perfusion. This delayed neuronal death is largely due to excessive calcium entry into neurons. For many years, therapeutic strategies have focused on the Ca2+-permeable glutamate receptor gated channels as the main target. However, efforts to prevent cell death through the therapeutic use of glutamate receptor antagonists have been disappointing. Although intolerance of glutamate receptor antagonists and timing issues largely contribute to the failure of clinical trials, an emerging new concept also favors the idea that other sources of Ca2+ entry might be equally important in determining the ischemic neuronal death. We have now strong evidence that oxidative stress and oxygen free radicals, produced in ischemia, activate a novel Ca2*-permeable cation channel (the free radical activated channel: FRAC) in cortical neurons and our preliminary data demonstrated that this channel is likely responsible for a glutamate-independent delayed Ca2+ overload in ischemic neuronal death. Our objective is to fully characterize the ionic properties, pharmacology profile and the regulation of this channel. We will attempt to determine the specific form of free radicals responsible for the channel activation and the mechanism of how free radicals activate the FRAC. Simultaneous Ca2+imaging and patch-clamp recording will be used to quantify the Ca2+ entry through FRACs. We will also determine how free radicals alter the excitability of acute! dissociated neurons and the neurons in hippocampal slices. Finally, using in vitro and eventually in vivo ischemic models, we will determine if preventing the activation of FRACs protects neurons from ischemic death.General Hypothesis: Activation of a Ca2+-permeable cation channel by oxygen free radicals (the FRAC) is responsible, at least partially, for a glutamate-independent, delayed Ca2+ overload in ischemic neuronal death.Specific Aims:(1). Electrophysiological Characterization of FRACs.(2). Pharmacological Characterization and intracellular Regulation of FRA Cs.(3). Fluorescent imaging Study of Ca2+ Response Induced by the FRACActivation.(4). Potential Role of FRACActivation in Glutamate-independent Ischemic Neuronal InjuryOur long-term objective is to identify new targets, in addition to glutamate receptors, and novel strategies to protect brain cells from the damage that accompanies the stroke.