Structural and functional role of the extracellular loops and C-terminal domain of the voltage-gated sodium ion channel

Voltage-gated sodium ion channels (Nav) are central to action potential initiation through regulating the entry of sodium ions (Na+) into excitable cells including cardiomyocytes and neurones. The α-subunit of Nav consists of four homologous domains (DI-DIV), each consisting of six transmembrane helices (S1-S6). Helices S5-S6 of each domain forms the lining of the central pore through which sodium ions (Na+) enters the cell upon channel activation. Helices S1-S4 of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential. Additionally, Nav channels include an extracellular turret region, whose role in channel function is poorly understood. The C-terminal domain (CTD) of Nav, connected to the DIV-S6, interacts with various other proteins including calmodulin (CaM) and fibroblast growth factor (FGF13) and mediates various regulatory roles. The subtype Nav1.5 is primarily expressed in the heart where it initiates the cardiac action potential whereas Nav1.7 is found in the peripheral nervous system where it is associated with nociception. Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms. Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Habib, Zaki

Advisors dc:contributor.advisor

• Huang, Christopher
• Jackson, Antony

• Ion Channel
• Channelopathy
• scFv phage display
• Protein-protein interaction
• Recombinant protein production and purification
• Molecular biology
• Electrophysiology

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.89428

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/342011