Abstract 11526: Voltage Gated Sodium Channel β1 Subunit Mimetic Peptide PS2L Increases Regulated Intramembrane Proteolysis of SCN1B/β1

Abstract

Clinical treatment for cardiac arrhythmias based on targeting ion channels is limited and can have deadly side effects. We previously reported that acute administration of SCN1B mimetic peptide βadp1 resulted in decreased action potential conduction velocity and increased arrhythmias in isolated guinea pig hearts. These changes were associated with disruption of trans-homophilic adhesion of the β1 subunit within gap junction-adjacent perinexal domains in intercalated discs.Following a strategy reported by two other independent groups working on intercellular adhesion molecules containing Ig domains, N-cadherin and desmoglein-2, we dimerized a c-terminal βadp1 sequence resulting in the peptide PS2L.Using Electric Cell-Substrate Impedance Sensing (ECIS) with Chinese Hamster 1610 cells stably expressing β1, we found that PS2L increases β1 mediated adhesion not only acutely, but unexpectedly for over 48 hours post-treatment. Regulated Intramembrane Proteolysis (RIP) of β1 sequentially by BACE-1 and γ-secretase results in a cleaved intracellular fragment that translocates to the nucleus and alters transcription of various genes, including VGSC subunits. Therefore, we hypothesized the long-term gain-of-adhesion-function with PS2L treatment resulted from increased RIP of β1, with downstream VGSC gene expression effects. Inhibition of γ-secretase with DAPT accumulates a C-terminal fragment (CTF) of β1 during RIP, enabling quantification of changes to RIP. Thus, 1610β1 cells were treated with 50μM PS2L, 1μM DAPT, 50μM PS2L+1μM DAPT or 0.1% DMSO control. Cells were fixed for β1 immunofluorescence (IF) or lysate was collected at 6-, 24-, and 48-hours post treatments for Western blotting.IF showed an increase in abundance of β1 48 hours after βadp1 treatment compared to DMSO, especially at cell borders. DAPT treatment significantly increased CTF levels compared to control at all timepoints (p<.0001). However, PS2L+DAPT treatment resulted in greater accumulation of β1 CTF levels over DAPT alone at the 6-hour timepoint but not later. (n=3, p<.05) In sum, our data indicates that PS2L may upregulate RIP of β1 with potential downstream effects on gene transcription of VGSC subunits, suggesting a path to a novel class of antiarrhythmics.