Converting the sodium-potassium pump into a shutdown channel

将钠钾泵转变为关闭通道

• 批准号: RGPIN-2017-04624
• 负责人: Andrew, Robert
• 金额: $ 1.89万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=681413
• 关键词: Converting; sodium; potassium; pump; into

Central neurons undergo spreading depolarization (SD) which shuts down brain function during acute metabolic demand. SD migrates across gray matter at 1-5 mm/min and has evolved in insects (and perhaps in the vertebrates) to induce brain silence under the stress of anoxia, head injury or sudden temperature change. SD is behaviorally protective by reducing movement but can kill neurons if it lasts many minutes. SD immediately follows failure of the Na/K ATPase but the molecular mechanism linking it the massive inward current driving SD is unknown and elusive. Blockade of voltage- or ligand-gated channels does not prevent the SD-like `anoxic` depolarization (AD). The molecular action the marine poison (palytoxin, Ptox) is known to specifically bind the Na/K pump at picomolar amounts, converting it from an ATP-requiring transporter to an open cationic channel. The sudden Na+ influx and K+ efflux we show emulates SD at a mere 10-100 nM in live brain slices of mouse or rat. Our hypothesis is that severe metabolic stress likewise converts the Na/K ATPase into a channel that drives SD, evoking the neuronal shutdown that is prevalent across animal classes. Thus the molecular action of Ptox can provide insight to CNS shutdown.*** The proposal has 3 Objectives: 1) Show with membrane patch recording by our HQP Peter G. that the Na/K ATPase opens to drive SD in amphibian and mammalian neurons of the higher brain. Direct demonstration of pump conversion to a channel requires recording across membrane patches from neurons. A Ptox-evoked 12 pS single channel conductance represents opening of a single pump transporter. 2) Demonstrate that conversion from pump to channel also drives SD in locust and Drosophilia where the molecular details of SD can be assessed in a large cohort using genetically variable strains. With the expertise of HQP Dr. Kristin S. we will extend patch experiments to insect brain. 3) Our pilot data from rat show SD is evoked by rapid temperature shifts to 40 or to 13oC, similar to heat- and chill-coma in insects. We will study brain slices from homeotherms under variable temperature, simulating poikilothermy to directly compare with naturally cold-blooded animals such as frog and insect.*** Shutdown elicited by SD reduces oxidative injury and bypasses partial depolarization that drives epileptiform/spastic activity. As well SD inactivates both Na+ channels...and the animal itself. But in homeotherms this immediate behavioral protection lasts only minutes and quickly leads to neuronal death, a process we do not understand. My Canadian lab together with my 3 HQPs represent the first neuroscience facility to directly study the CNS Na/K pump and the channels that open as it fails. We will identify the SD channel by showing a) it activates under metabolic stress; b) it is not blocked by inhibitors of standard channels across animal species; and c) it may share properties similar to the pump channel induced by Ptox.

中枢神经元经历扩散去极化（SD），在急性代谢需求期间关闭大脑功能。 SD 以 1-5 毫米/分钟的速度穿过灰质，并在昆虫（或许还有脊椎动物）中进化，在缺氧、头部受伤或温度突然变化的压力下诱导大脑沉默。 SD 通过减少运动来提供行为保护，但如果持续几分钟，就会杀死神经元。 SD 紧随 Na/K ATP 酶失效而发生，但将其与驱动 SD 的大量内向电流联系起来的分子机制尚不清楚且难以捉摸。电压门控通道或配体门控通道的阻断不会阻止类似 SD 的“缺氧”去极化 (AD)。已知海洋毒物（海藻毒素，Ptox）的分子作用以皮摩尔量特异性结合 Na/K 泵，将其从需要 ATP 的转运蛋白转化为开放的阳离子通道。我们展示的 Na+ 突然流入和 K+ 流出模拟了小鼠或大鼠活体脑切片中仅 10-100 nM 的 SD。我们的假设是，严重的代谢应激同样会将 Na/K ATP 酶转化为驱动 SD 的通道，从而引起动物类别中普遍存在的神经元关闭。因此，Ptox 的分子作用可以为 CNS 关闭提供见解。*** 该提案有 3 个目标：1) 通过我们的 HQP Peter G. 的膜片记录显示 Na/K ATP 酶打开以驱动两栖动物和哺乳动物高级大脑神经元中的 SD。直接演示泵向通道的转换需要跨神经元的膜片进行记录。 Ptox 诱发的 12 pS 单通道电导代表单泵转运蛋白的打开。 2) 证明从泵到通道的转换也驱动了蝗虫和果蝇中的 SD，其中可以使用遗传变异菌株在一个大队列中评估 SD 的分子细节。凭借 HQP Kristin S. 博士的专业知识，我们将把贴片实验扩展到昆虫大脑。 3) 我们来自大鼠的试验数据显示，SD 是由温度快速变化至 40 或 13°C 引起的，类似于昆虫的热昏迷和冷昏迷。我们将研究不同温度下恒温动物的脑切片，模拟变温动物，直接与青蛙和昆虫等自然冷血动物进行比较。*** SD 引起的关闭可减少氧化损伤，并绕过导致癫痫样/痉挛活动的部分去极化。 SD 也会使 Na+ 通道和动物本身失活。但在恒温动物中，这种直接的行为保护只持续几分钟，并很快导致神经元死亡，这是一个我们不了解的过程。我的加拿大实验室和我的 3 个 HQP 代表了第一个直接研究 CNS Na/K 泵及其故障时打开的通道的神经科学机构。我们将通过显示 a) SD 通道在代谢压力下激活来识别 SD 通道； b) 它不会被跨动物物种的标准通道抑制剂阻断； c) 它可能具有与 Ptox 引起的泵浦通道类似的特性。