Abstract：
In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm ) reaches the threshold for activation of the voltage-gated Na+ channels (VGNCs) Nav 1.2 and Nav 1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na+ , Vm and Ca2+ imaging in combination with partial block of Nav 1.2 by the peptide G1 G4 -huwentoxin-IV, we demonstrate an exclusive role of Nav 1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Nav 1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca2+ -activated K+ channels (CAKCs). Indeed, Ca2+ influx via Nav 1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9-anhydrotetrodotoxin, a moderately selective inhibitor of Nav 1.6, we concluded that the Ca2+ influx shaping the early phase of the AP is exclusive of Nav 1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav 1.2 reported here is important for understanding the physiology and pathology of neuronal excitability. KEY POINTS: We optically analysed the action potential generated in the axon initial segment of mouse layer-5 neocortical pyramidal neurons and its associated Na+ and Ca2+ currents using ultrafast imaging techniques. We found that partial selective block of the voltage-gated Na+ channel Nav 1.2, produced by a recently developed peptide, widens the shape of the action potential in the distal part of the axon initial segment. We demonstrate that this effect is due to a reduction of the Ca2+ influx through Nav 1.2 that activates BK Ca2+ -activated K+ channels. To validate our conclusions, we generated a neuron model that reproduces the ensemble of our experimental results. The present results indicate a specific role of Nav 1.2 in the axon initial segment for shaping of the action potential during its generation.
摘要：
在新皮质层-5锥体神经元中，当膜电位（Vm）达到电压门控Na通道（VGNC）Nav1.2和Nav1.6激活的阈值时，在轴突初始片段（AIS）中产生动作电位（AP）。然而，尽管已知这些VGNC在AIS的空间分布和生物物理特性上有所不同，对两种渠道之间功能差异的理解仍然难以捉摸。这里,使用超快Na+，Vm和Ca2+成像结合肽G1G4-huwentoxin-IV部分阻断Nav1.2，我们展示了Nav1.2在塑造生成AP方面的独家作用。准确地说，我们显示，Nav1.2的选择性阻断〜30％加宽AIS远端部分的AP，并且我们证明这种效应是由于BKCa2激活的K通道（CAKCs）的激活丧失。的确,通过Nav1.2流入的Ca2会激活BKCAKCs，从而确定AIS中AP的振幅和复极化的早期阶段。通过使用4,9-脱水河豚毒素的对照实验，这是Nav1.6的中等选择性抑制剂，我们得出结论，形成AP早期阶段的Ca2流入不包括Nav1.2。因此，我们用神经元模型模拟了这个结果，其中测试的不同离子通道的作用再现了实验证据。本文报道的Nav1.2的独特作用对于理解神经元兴奋性的生理学和病理学很重要。关键点：我们使用超快成像技术光学分析了小鼠5层新皮质锥体神经元轴突初始部分产生的动作电位及其相关的Na+和Ca2+电流。我们发现，由最近开发的肽产生的电压门控Na通道Nav1.2的部分选择性阻断，扩展轴突初始段远端部分的动作电位的形状。我们证明了这种作用是由于通过Nav1.2的Ca2流入减少，从而激活了BKCa2激活的K通道。为了验证我们的结论，我们生成了一个神经元模型，它再现了我们实验结果的集合。目前的结果表明，Nav1.2在轴突初始段中对动作电位的形成具有特定的作用。摘要图例在新皮质锥体神经元的轴突初始部分，电压门控Na通道Nav1.2通过提供Na电流和Ca2电流来激活BKCa2激活的K通道，从而形成动作电位的动力学。本文受版权保护。保留所有权利。
