A novel cation channel in excitatory neuronal injury

说明(申请人提供)：钙是中枢神经系统中最重要的离子之一，对调节神经元兴奋性、突触传递、神经元发育和分化至关重要。钙稳态的改变已被证明参与了各种神经系统疾病/紊乱的病理过程。例如，细胞内Ca~(2+)蓄积([Ca~(2+)]_i)被认为是脑缺血的主要病理特征。在[Ca~(2+)]i升高的同时，缺血还导致细胞外Ca~(2+)浓度([Ca~(2+)]_e)的急剧下降。虽然众所周知，[Ca~(2+)]_j的升高在兴奋性神经元损伤中起关键作用，但[Ca~(2+)]_e的改变是否在脑缺血的病理过程中起作用尚不清楚。我们先前已经证明，将[Ca~(2+)]e降低到脑缺血时常见的水平，通过激活非选择性阳离子通道，强烈地去极化和兴奋培养的海马神经元。该通道具有不同于已知通道的电生理特性和药理特性，提示激活了一种新的钙敏感离子通道。我们的初步研究还表明，该通道的激活导致[Ca~(2+)]i升高，并增强了NMDA受体介导的膜反应和神经元损伤。因此，我们假设，脑缺血时[Ca~(2+)]e降低到一定水平，可激活一种独特的钙敏感非选择性阳离子通道(CsNSC)。这些通道的激活诱导膜去极化和神经元兴奋，从而直接或间接地通过增强NMDA受体介导的反应而导致兴奋性毒性。我们的目的是提供更多证据，证明csNSC确实是一种独特的新通道，并探讨其在缺氧/缺血性神经元损伤中的病理作用。除了培养的神经元，我们还将在急性分离的成熟神经元和脑片中表征csNSC通道的详细电生理特性，开发药理学特征并寻找特定的通道阻滞剂。我们将定义csNSC通道的钙通透性，并确定缺血处理是否增强了钙通透性。由于NMDA通道在兴奋性神经元损伤中起关键作用，我们将详细描述csNSC通道与NMDA受体介导的膜反应和神经元损伤的相互作用。利用体外缺血模型，我们将确定阻止csNSC通道的激活是否能保护神经元免受缺血损伤。具体目标是：1.提供进一步的证据表明，将[钙]e降低到脑缺血时的水平，可以激活中枢神经系统中一个独特的非选择性阳离子通道。2.证明了csNSC通道具有钙离子通透性。3.证实csNSC激活可增强NMDA通道功能。4、确定csNSC通道在缺血性神经元损伤中的潜在作用。

DESCRIPTION (provided by applicant): Calcium is one of the most important ions in the central nervous system, essential for the regulation of neuronal excitability, synaptic transmission, neuronal development and differentiation. Alterations of Ca2+ homeostasis have been shown to be involved in the pathology of various neurological diseases/disorders. Accumulation of intracellular Ca2+ ([Ca2+]i), for example, has been recognized as a central pathological feature in brain ischemia. Along with an increase in [Ca2+]i, ischemia also causes a dramatic decrease in the concentration of extracellular Ca2+ ([Ca2+]e). Although it is well-known that the increase of [Ca2+]j is critical for excitatory neuronal injury, it is not clear whether the alteration of [Ca2+]e plays any role in the pathology of brain ischemia. We have previously demonstrated that lowering [Ca2+]e to the level commonly seen in brain ischemia strongly depolarized and excited cultured hippocampal neurons through activation of a non-selective cation channel. This channel has electrophysiological properties and pharmacological profiles different from known channels, suggesting activation of a novel Ca2+-sensitive ion channel. Our preliminary study also demonstrated that activation of this channel caused an increase in [Ca2+]i and potentiated NMDA-receptor mediated membrane responses as well as neuronal injury. We therefore hypothesize that decreases of [Ca2+]e to the level seen in brain ischemia activates a distinct Ca2+-sensing non-selective cation (csNSC) channel. Activation of these channels induces membrane depolarization and neuronal excitation, which contributes to excitotoxicity either directly, or indirectly through potentiation of NMDA receptor mediated responses. Our objective is to provide additional evidence that csNSC is indeed a distinct new channel and to investigate its pathological role in hypoxic/ischemic neuronal injury. In addition to cultured neurons, we will characterize detailed electrophysiological properties of the csNSC channel in acutely dissociated mature neurons and the neurons in brain slices, develop a pharmacological profile and search for a specific channel blocker. We will define the Ca2+-permeability of the csNSC channel, and determine whether ischemic treatment enhances the Ca2+-permeability. Since NMDA channels play a critical role in excitatory neuronal injury, we will characterize detailed interaction of csNSC channels with NMDA receptor-mediated membrane responses and neuronal injury. Using in vitro ischemic models, we will determine whether preventing the activation of csNSC channels protects neurons from ischemic injury. Specific Aims are: 1. Provide further evidence that lowering [Ca2+]e to the level seen in brain ischemia activates a distinct nonselective cation channel in the central nervous system. 2. Demonstrate that csNSC channels are Ca2+-permeable. 3. Demonstrate potentiation of NMDA channel function by csNSC activation. 4; Determine the potential role of csNSC channels in ischemic neuronal injury.