Proteolytic processing of sodium channel beta1 subunits

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.