Studies on voltage-gated sodium channel β3 subunit structure and cancer-related functions using single-chain variable fragment antibodies and bioinformatics

Voltage-gated sodium channels (VGSC), embedded in the plasma membrane of cells, play a pivotal role in generating sodium currents and action potentials. The mammalian VGSCs consist of a large pseudo-tetrameric pore-forming α subunit, the channel protein, which associates with one or more β-subunits. In humans, nine types of VGSC Nav1 channel α subunit isoforms (Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) and four types of β subunit isoforms (β1, β2, β3, and β4) (gene names: *SCN1-11A* and *SCN1-4B*) have been identified in various tissues. This thesis describes the use of single-chain variable fragment (scFv) antibodies to study the binding site of the β3 subunit on the pain-sensing channel, Nav1.7, and investigate the role of the VGSC β3 subunit in cancer guided by bioinformatic analysis. *In silico* docking was used to construct a binding model between the Nav1.7 α subunit and β3 subunit, and molecular dynamic simulations were employed to assess the binding stability of this model. These computational structural analyses provide an *in silico* model that was validated in subsequent scFv mapping experiments. Using scFvs that specifically recognized distinct regions of Nav1.7, the binding site of the β3 subunit on the Nav1.7 α subunit was identified. The results substantiate that the β3 subunit binds to the Nav1.7 α subunit in a manner akin to the β1 subunit. This study offers a valuable strategy for studying the extracellular domain of plasma membrane complexes under cellular conditions, complementing cryo-electron microscopy (cryo-EM) and X-ray crystallography approaches. In the context of cancer research, the systematic literature review and bioinformatics analysis indicated that the expression of the VGSC β3 subunit gene (*SCN3B*) is associated with reduced glioma severity and regulated glioma immunity and migration. Subsequently, the effect of the β3 subunit on glioma cell migration was experimentally investigated. The results reveal that the β3 subunit inhibits glioma cell motility via the β3 subunit immunoglobulin (β3 Ig) domain and this function is not reliant on chemo-sensing. Instead, the β3 Ig domain governs actin-based cell protrusion, reducing network actin (lamellipodia and membrane ruffle) while augmenting bundle actin (filopodia) in glioma cells. Finally, through immunoprecipitation and mass spectrometry, several pathways underlying the regulatory function of the β3 subunit in glioma cell actin organization were identified. In conclusion, this study advances our understanding of the structure and significance of the VGSC β3 subunit in cancer biology.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Liu, Hengrui

Advisor dc:contributor.advisor

• Jackson, Antony

• Bioinformatics
• cancer
• scFv
• SCN3B
• structure
• VGSC

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/10d695d1-7197-41c3-ac69-48f544dbe2af/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.107264

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/366282