Proteolytic processing of sodium channel beta1 subunits

电压门控钠离子通道(VGSCs)负责电兴奋细胞(包括神经元和肌肉细胞)的动作电位。VGSC含有一个成孔的α亚基和一个或两个辅助的β亚基。β亚基调节通道活性，在其他离子通道辅助亚基中是独一无二的，因为它们也是细胞黏附分子。因此，VGSC不仅是离子通道，而且是细胞黏附复合体。特别是，在中枢神经系统发育过程中，β1亚基调控动作电位的激发、轴突的生长和神经元的迁移。有趣的是，Beta1也在乳腺癌细胞中表达，在那里它调节细胞的迁移、侵袭和转移。VGSCβ亚基含有参与阿尔茨海默病病理的分泌酶进行处理的裂解位点。分泌酶活性也正在成为乳腺癌的一个重要靶点。然而，Beta1的蛋白分解过程的功能后果尚不清楚。这个项目的目的是验证这样的假设，即β1亚基被分泌酶切割，并调节黏附、轴突生长、细胞迁移和电活动。我们将使用一系列复杂的系综和单分子显微镜方法，例如共聚焦显微镜、TIRF显微镜、FRAP来探索神经元和乳腺癌细胞中β1亚单位的化学计量和循环。我们将使用药物并通过产生切割位点已被修饰的Beta1突变体来调节分泌酶的活性。我们将使用染色质免疫沉淀(CHIP)等分子方法研究Beta1处理对基因表达的功能影响。重要的是，我们将利用细胞迁移分析和全细胞膜片钳电生理记录来研究蛋白质降解处理对细胞迁移和通道功能的影响。因此，该项目将使学生接触到该领域领先的实验室中的一系列尖端细胞生物学技术。由于Beta1在大脑发育和许多疾病中发挥关键作用，该项目有望为潜在的治疗靶点提供新的机制见解。

Voltage-gated Na+ channels (VGSCs) are responsible for action potentials in electrically excitable cells, including neurons and muscle cells. VGSCs contain a pore-forming alpha subunit with one or two auxiliary beta subunits. The beta subunits regulate channel activity and are unique amongst other ion channel auxiliary subunits because they are also cell adhesion molecules. VGSCs are therefore not only ion channels, but also cell adhesion complexes. In particular, the beta1 subunit regulates action potential firing, neurite outgrowth and neuronal migration during central nervous system development. Interestingly, beta1 is also expressed in breast cancer cells, where it regulates cellular migration, invasion and metastasis. VGSC beta subunits contain cleavage sites for processing by secretases that are involved in Alzheimer's disease pathology. Secretase activity is also emerging as an important target in breast cancer. However, the functional consequences of proteolytic processing of beta1 are not understood. The aim of this project is to test the hypothesis that beta1 subunits are cleaved by secretases and that this regulates adhesion, neurite outgrowth, cellular migration and electrical activity. We will use a range of sophisticated ensemble and single-molecule microscopy approaches, e.g. confocal microscopy, TIRF microscopy, FRAP to explore the stoichiometry and cycling of beta1 subunits, in neurons and breast cancer cells. We will modulate secretase activity using drugs and by generating beta1 mutants in which the cleavage sites have been modified. We will study the functional consequences of beta1 processing on gene expression using molecular approaches such as chromatin immunoprecipitation (ChIP). Importantly, we will study the effect of proteolytic processing on cellular migration and channel function using cell migration assays and whole cell patch clamp electrophysiological recording. The project will therefore expose the student to a range of cutting-edge cell biology techniques in labs that are leading in this field. As beta1 plays a key role in brain development and in a number of diseases, this project is expected to provide novel mechanistic insights into a potential therapeutic target.