The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K⁺ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

内侧哈贝拉（MHb）接收间隔输入并将传出物发送至足突间核，并与压力，抑郁，记忆和尼古丁戒断综合征有关。我们先前通过免疫荧光显微镜检查发现，细胞黏附分子nectin-2α在发育中和成年小鼠MHb中的胆碱能神经元中表达，并位于簇状胆碱能神经元的相邻躯体之间的边界处，其中电压门控A型K ⁺通道Kv4.2已本地化。我们通过免疫电子显微镜进一步表明，Kv4.2位于膜特化处（MSs），而nectin-2α主要位于这些MS外。此外，我们证明了nectin-2的遗传消融延迟了Kv4.2在正在发展的MHb中MS的定位。我们在这里研究了nectin-2α如何调节MS上Kv4.2的这种定位。体外生化分析表明，nectin-2α与Kv4.2二肽基氨基肽酶样蛋白6（DPP6）的辅助蛋白相互作用，但不与Kv4.2或另一种辅助蛋白Kv通道相互作用蛋白1（KChIP1）相互作用。免疫荧光显微镜分析表明，DPP6与nectin-2α共定位在发育中和成年MHbs的聚集胆碱能神经元的相邻躯体之间的边界处。在该边界上的免疫电子显微镜分析表明，DPP6位于MS的内部和外部。Nectin-2的遗传消融并没有影响DPP6在发育中和成年MHb中聚集的胆碱能神经元的相邻躯体之间的边界处的定位。这些结果表明，nectin-2α与DPP6相互作用，但以不依赖DPP6的方式调节MS上Kv4.2的定位。