Dynamics of Kv1 channel transport in axons.

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K+ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons.

沿轴突精确靶向的各种离子通道的协同作用对于动作电位的起始和传播以及神经递质的释放至关重要。然而，轴突中通道蛋白运输的动力学仍然未知。在此，通过延时成像，我们发现荧光标记的Kv1.2电压门控K⁺通道（YFP - Kv1.2）在海马轴突中以离散的点状结构双向移动。表达Kvβ2（一种Kv1辅助亚基）显著增加了这些点状结构在顺行和逆行方向上的速度、移动距离以及移动时间百分比。通过小干扰RNA（siRNA）抑制与Kvβ2相关的蛋白，即正端结合蛋白EB1或驱动蛋白II/KIF3A，显著降低了YFP - Kv1.2点状结构在两个方向上的移动速度。Kvβ2突变体，无论是Kv1.2 - Kvβ2结合还是Kvβ2 - EB1结合被破坏，都无法增加YFP - Kv1.2点状结构的速度，这证实了Kvβ2的核心作用。此外，荧光标记的Kv1.2和Kvβ2沿轴突共同移动。令人惊讶的是，当与Kv1.2和Kvβ2共同移动时，EB1的移动速度似乎明显快于其正端追踪速度。最后，利用表达YFP融合蛋白的裂殖酵母粟酒裂殖酵母作为参考标准来校准我们的显微镜，我们估算了轴突点状结构中YFP - Kv1.2四聚体的数量。综上所述，我们的结果表明，适量的Kv1通道及其相关蛋白是Kv1通道蛋白沿轴突高效运输所必需的。