Proteolytic processing of sodium channel beta1 subunits

Lead Research Organisation: University of York
Department Name: Biology

Abstract

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.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1643216 Studentship BB/M011151/1 30/09/2015 29/09/2019 Alexander Haworth
 
Description My research focuses on one protein, scn1b, and its functional regulation by a family of enzymes known as secretases. Scn1b is a subunit of voltage-gated sodium channels and its expression enhances the magnitude of the inward sodium current. Initially, my work focused on developing a range of cDNA constructs encoding scn1b mutants that model the effect of secretase cleavage. For instance, one construct encoded a form of scn1b resistant to secretase cleavage, while another construct encoded the scn1b intracellular domain, the secretase cleavage product of scn1b. Using these constructs, I demonstrated that the intracellular domain of scn1b is required for sodium current enhancement, however secretase cleavage and intracellular domain formation is not required for this process.
Exploitation Route The functional impact of secretase cleavage on scn1b is still unreported. My work suggests secretases are not involved in sodium current regulation, which raises the question of what is the purpose of secretase cleavage of scn1b? As scn1b misexpression is implicated in epilepsy, neurodevelopmental conditions and cancer, understanding the impact of secretase cleavage may provide further knowledge on the role of scn1b in these disorders.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description PhD Facilities Award
Amount £3,900 (GBP)
Organisation University of York 
Sector Academic/University
Country United Kingdom
Start 07/2017 
End 08/2018