Converting the sodium-potassium pump into a shutdown channel

中枢神经元经历扩散性去极化(SD)，在急性代谢需求时关闭大脑功能。SD以1-5 mm/min的速度在灰质中迁移，并在昆虫(可能也包括脊椎动物)中进化，在缺氧、头部损伤或突然温度变化的压力下导致大脑沉默。SD通过减少运动来保护行为，但如果持续许多分钟，就会杀死神经元。SD紧跟在Na/K ATPase失效之后，但将其与巨大的内向电流驱动SD联系起来的分子机制尚不清楚和难以捉摸。阻断电压或配基门控通道并不能阻止SD样的“缺氧”去极化(AD)。已知海洋毒素(Palyoxin，Ptox)的分子作用能以皮摩尔量与Na/K泵结合，将其从需要ATP的转运体转换为开放的阳离子通道。在小鼠或大鼠的活体脑片中，我们显示的突然的钠内流和钾外流仅在10-100 nm处模拟SD。我们的假设是，严重的代谢应激同样会将Na/K ATPase转化为驱动SD的通道，引发在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的关闭提供洞察力。 该提案有3个目标：1)由我们的HQP Peter G.用膜片记录显示，Na/K ATPase开放以驱动两栖动物和哺乳动物高等大脑神经元的SD。泵转换为通道的直接演示需要记录来自神经元的跨膜斑块。由PTOX引起的12ps单通道电导表示单泵传输器打开。2)证明了在蝗虫和果蝇中，从泵到通道的转换也驱动了SD，在这些地方，SD的分子细节可以使用遗传可变的菌株在一个大的队列中进行评估。在HQP Kristin S.博士的专业知识下，我们将把斑贴实验扩展到昆虫的大脑。3)我们在老鼠身上的实验数据显示，SD是由温度快速变化到40或13摄氏度引起的，类似于昆虫的热昏迷和冷昏迷。我们将研究变温下恒温动物的脑片，模拟变温，直接与青蛙、昆虫等自然冷血动物进行比较。 SD引起的停机可减少氧化损伤，并绕过导致癫痫样/痉挛活动的部分去极化。此外，SD使钠通道和动物本身都失活。但在恒温动物中，这种即时的行为保护只持续几分钟，很快就会导致神经元死亡，这是我们不了解的过程。我的加拿大实验室和我的三个HQP代表着第一个直接研究CNS Na/K泵和失效时打开的通道的神经科学机构。我们将通过显示a)它在新陈代谢压力下被激活；b)它不被跨动物种类的标准通道的抑制剂阻断；c)它可能具有类似于由Ptox诱导的泵通道的性质来识别SD通道。

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.