Function of TRAAK current in the rat node of Ranvier

我们提出实验来证明力学敏感TRAAK通道在大鼠Ranvier结中的新功能。在哺乳动物的Ranvier节点中，动作电位是由Na+电流激活和失活引起的，电压依赖性K通道的激活不参与复极。这与动作电位的经典离子理论相反，在动作电位理论中，电压相关的K电流的激活对复极化很重要。哺乳动物节点的外向电流是由相对较大的电压无关的漏电流提供的（Schwarz and Eikhof, 1987; Schwarz et al., 1995）。最近，R. MacKinnon （Brohawn et al., 2012）对人类TRAAK通道进行了结晶化。麦金农实验室还发现，TRAAK通道几乎完全局限于兰维耶结，它们不会出现在神经系统的其他地方。在提出的实验中，我们想分析这些新发现的力学敏感节点TRAAK通道的功能。该分析的一个重要先决条件是检测TRAAK阻断化合物（Su et al., 2016）。我们使用Nonner钳（Nonner 1969）对单髓大鼠神经纤维的Ranvier节点进行了初步实验。大约30%的“泄漏”电流被TRAAK通道阻滞剂阻断，10 μ M花生四烯酸和10 mM三氯乙醇的“泄漏”电流振幅几乎翻了一番。由于这些发现是TRAAK通道的特征，它们表明TRAAK通道存在于Ranvier淋巴结。我们还表明，通过用低渗溶液对所研究的节点进行超压，拉伸轴浆，可以激活节点TRAAK电流。在我们的拨款申请中，我们提出了实验来验证这样的假设，即在动作电位上升期间，Na+和水的流入引起的短暂节肿胀激活了机械敏感的TRAAK通道。提出TRAAK的功能是拉伸激活的K+电流会加速动作电位复极化。我们想记录TRAAK阻滞剂应用前后的动作电位，并以动作电位为电位模板进行动作电位钳夹实验，观察TRAAK阻滞剂是否使内向电流变宽。由于TRAAK通道对温度非常敏感，我们希望在生理温度下进行实验。我们预计漏电流的TRAAK电流分量会大大增加。如果我们的假设是正确的，那么我们将发现一种新的机制来稳定节点动作电位的产生。

We propose experiments to prove a new function of mechano-sensitive TRAAK channels in the rat node of Ranvier. In the mammalian node of Ranvier the action potential is brought about by Na+ current activation and inactivation, activation of voltage-dependent K channels does not contribute to repolarization. This is in contrast to the classical ion theory of the action potential where activation of voltage-dependent K current is important for repolarization. In the mammalian node outward current is provided by a relatively large voltage-independent leakage current (Schwarz and Eikhof, 1987; Schwarz et al., 1995). Recently, the human TRAAK channel was crystallized by R. MacKinnon (Brohawn et al., 2012). The MacKinnon lab also found that TRAAK channels are localized almost exclusively in the node of Ranvier, they do not occur elsewhere in the nervous system. In the proposed experiments we want to analyze the function of these newly discovered mechano-sensitive nodal TRAAK channels. An important prerequisite for this analysis is the detection of a TRAAK blocking compound (Su et al., 2016). We have done preliminary experiments in nodes of Ranvier of single myelinated rat nerve fibers by using the Nonner clamp (Nonner 1969). About 30% of the "leakage" current was blocked by the TRAAK channel blocker and the amplitude of the "leakage" current was almost doubled by 10 µM arachidonic acid and 10 mM trichlorethanol. Since these findings are characteristic for TRAAK channels they indicate the presence of TRAAK channels in the node of Ranvier. We have also shown that nodal TRAAK current is activated by stretching the axoplasma by superfusing the node under investigation with a hypotonic solution. In our grant application we propose experiments to test the hypothesis that the mechano-sensitive TRAAK channels are activated by the transient nodal swelling induced by the influx of Na+ and water during the upstroke of the action potential. The proposed function of TRAAK would be that the stretch-activated K+ current would accelerate action potential repolarization. We want to record action potentials before and after application of the TRAAK blocker and we want to carry out action potential clamp experiments with an action potential as potential template to see whether the TRAAK blocker broadens the inward current. Since TRAAK channels are very temperature-sensitive we want to do experiments at physiological temperature. We expect that the TRAAK current component of the leakage current increases considerably. If our hypothesis is correct then we would have discovered a new mechanism to stabilize the generation of the nodal action potential.