Caspr与ATP1B3互作调控钠钾泵装配和维持神经血管单元功能的机制

Na+/K+ pump, also called Na+/K+-ATPase, is essential for membrane potential recovery after depolarization and maintenance of neuron excitability. Na+/K+pump was composed by catalytic α-subunit and highly glycosylated β-subunit. The regulatory mechanism controls the assembly of Na+/K+pump onto plasma membrane remains unclear. Our preliminary results demonstrated that Caspr, a protein specifically expressed in neuron and brain endothelium, is able to bind with immature form of β3-subunit of Na+/K+ pump (ATP1B3) and enhance the glycosylation of ATP1B3. Knockdown of Caspr resulted in accelerated degradation of ATP1B3 as well as α-subunit of Na+/K+ pump, suggested that Caspr is a crucial regulator for accurate assembly of Na+/K+ pump. In this project, we attempt to study the molecular mechanism of how the interaction of Caspr with ATP1B3 regulates assembly of Na+/K+ pump, as well as its functional relevance in the activity of neural-vascular unit. In other words, we will investigate the mechanism of Caspr-ATP1B3 interaction regulates membrane translocation of α1β3 Na+/K+ pump on brain microvascular endothelial cells and α3β3 Na+/K+ pump on neurons. Furthermore, the conditional knockout mice will be utilized to explore the coordination of Caspr-ATP1B3 interaction regulates membrane assembly of Na+/K+ pump in the functional activity of neural-vascular unit. This project is dedicated to elucidate the assembly mechanism of Na+/K+ pump and provide new theoretical basis for the function of Na+/K+ pump in neural activity.

脑组织钠钾泵对恢复去极化后神经元膜潜能、维持神经元兴奋性起基础作用。钠钾泵主要由α催化亚基和高度糖基化的β亚基组成。控制钠钾泵组装到细胞膜上的因素是什么？迄今尚不清楚。我们前期发现，特异性表达于神经元和脑微血管内皮细胞的Caspr能够与未成熟型β3亚基（ATP1B3）结合、促进ATP1B3糖基化，Caspr的敲减导致ATP1B3和α亚基快速降解，这提示Caspr可能是调控钠钾泵装配的关键因子。本项目将进一步研究Caspr与ATP1B3互作调控钠钾泵装配的分子机制及其在神经血管单元功能活动中的意义，即研究Caspr-ATP1B3互作调控α1β3钠钾泵在脑微血管内皮细胞、α3β3在神经元的膜定位机制，结合选择性敲除Caspr转基因小鼠，探讨Caspr-ATP1B3互作调控的钠钾泵装配在神经血管单元功能活动中的协调作用机制。本项目将为阐明钠钾泵装配机制及钠钾泵在神经活动中的作用提供新的理论基础。

钠钾泵也称Na+/K+ATP酶，主要由α催化亚基（α1-α4）和高度糖基化的β亚基（β1-β3）组成，广泛分布于包括脑在内的机体组织细胞，它对恢复去极化后神经元膜潜能和维持神经元兴奋性起基础作用。但控制钠钾泵组装到细胞膜上的机制，迄今尚不清楚。本项目在我们前期结果提示的Caspr（contactin associated protein，也称Caspr1）可能是调控钠钾泵装配因子的基础上，主要研究了Caspr1与ATP1B3（β3）互作调控脑微血管内皮细胞和神经元钠钾泵装配的分子机制及其在神经血管单元功能活动中的意义。本项目获得了以下原创性发现：1）Caspr1与未糖基化的ATP1B3核心蛋白的互作，促进了ATP1B3的糖基化加工成熟并维持了ATP1B3（β3亚基）和α1亚基的稳定性，进而促进了Na+/K+-ATP酶α1β3在脑微血管内皮细胞的膜定位，调控了依赖α1β3的脑内神经递质谷氨酸向血液的转运；2）神经元中的Caspr1通过与钠钾泵ATP1B3核心蛋白互作维持了突触部位钠钾泵α1、α3亚基的分布；3）神经元中Caspr1的敲降导致ATP1B3降解和突触部位钠钾泵膜定位的抑制，进而提高了神经元静息膜电位，增加了神经元兴奋性，促使钙离子内流增加，诱发突触囊泡过度释放；4）发现神经元Caspr1条件性敲除小鼠表现为抗焦虑行为，并揭示海马神经元突触传递增强可能是导致小鼠抗焦虑行为的原因之一。这些结果为阐明钠钾泵装配机制及钠钾泵在神经活动中的作用提供了新的理论基础。