Investigating the Cellular Mechanisms Underlying Nuclear Translocation of the Sodium Channel beta-1 Subunit Intracellular Domain
Abstract number :
1.48
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2024
Submission ID :
1455
Source :
www.aesnet.org
Presentation date :
12/7/2024 12:00:00 AM
Published date :
Authors :
Presenting Author: Sebastian Casillas-Lopez, – University of Michigan-Ann Arbor
Dyke McEwen, PhD – University of Michigan-Ann Arbor
Lori Isom, PhD – University of Michigan-Ann Arbor
Rationale: β1, encoded by the SCN1B gene, is a non-pore-forming subunit of the voltage-gated sodium channel (VGSC). β1 subunits are multi-functional proteins that regulate VGSC α-subunit cell surface density, channel kinetics, and participate in cell-cell and cell-matrix adhesion to modulate neurite outgrowth, neuronal pathfinding, and axon fasciculation. Pathogenic SCN1B variants have been linked to disease, including developmental and epileptic encephalopathy and cardiac arrhythmia. One example is Dravet syndrome, a devastating developmental and epileptic encephalopathy, with patients having a significant increased risk of Sudden Unexpected Death in Epilepsy (SUDEP). β1 subunits are substrates for regulated intramembrane proteolysis (RIP) by β-secretase 1, releasing the β1 extracellular domain, followed by γ-secretase cleavage to release the β1 intracellular domain (β1-ICD). The β1-ICD then traffics to the nucleus where it can alter mRNA expression levels, particularly SCN1A, the gene encoding Nav1.1. However, it remains unclear what mechanisms underlie β1-ICD trafficking to the nucleus. The goal of the current study is to identify the trafficking mechanisms that facilitate β1-ICD nuclear translocation following RIP.
Methods: A combined approach of immunocytochemistry (ICC), immunoprecipitation (IP), and western blot techniques were used to investigate mechanisms involved in β1-ICD translocation to the nucleus. Chinese Hamster Lung fibroblast cells (CHLs) expressing the β1-ICD were treated separately with two importin inhibitors, Ivermectin and Importazole, to investigate the role of the importin machinery in this process. IP and western blot from cell lines and mouse brain membranes were used to investigate whether β1 associates with the importin machinery in vitro and in vivo.
Results: ICC of CHLs expressing the β1-ICD treated with ivermectin or importazole showed that ivermectin treatment excluded the β1-ICD from the nucleus while importazole had no effect. This suggests that, while members of the importin protein family are likely involved in β1-ICD translocation, importin β1 may not play a role in β1-ICD trafficking to the nucleus. Further, our IP data support the finding that β1 is in complex with importin machinery.
Conclusions: Our data suggest that the β1-ICD translocates to the nucleus via an importin-dependent mechanism. Pharmacologic inhibition of these proteins, which prevents nuclear import, has begun to elucidate the importin proteins involved in β1-ICD trafficking to the nucleus. Future studies will further investigate the importin subtypes involved in β1-ICD nuclear translocation and subsequent gene regulation.
Funding: This study was funded by the AES BRIDGE Summer Internship Grant and NIH-R37NS076752 to LLI.
Basic Mechanisms