Development and Characterization of the Mode-of-Action of Inhibitory and Agonist Peptides Targeting the Voltage-Gated Sodium Channel SCN1B/β1 Subunit

Abstract

Treatment of cardiac arrhythmias by targeting ion channels is challenging, with safe and effective therapies remaining an unmet clinical need. Modeling and experimental studies have shown that a voltage-gated sodium channel (VGSC)-rich nanodomain at edge of the gap junction (GJ) called the perinexus could provide new mechanistic insights into normal and abnormal conduction of action potentials in the heart. We have reported that a 19 amino acid SCN1B (β1/β1B) mimetic peptide derived from the immunoglobulin domain of the VGSC subunit called βadp1 acutely disrupts β1-mediated adhesive interactions at cardiac perinexii, prompting arrhythmogenic changes during time courses of up to an hour. In the present study, we sought to gain further insight on βadp1 mode-of-action, as well as identifying new SCN1B (β1/β1B) mimetic peptides, with potential for inhibiting and/or promoting β1-mediated adhesion. This included studies of the effect of βadp1 and related peptides on SCN1B (β1/β1B) Regulated Intramembrane Proteolysis (RIP) - a signaling pathway that has been shown to effect gene transcription, including that of VGSC subunits. Using patch clamp to assay cell-cell contact-associated VGSC activity in cardiomyocytes, and electric cell substrate impedance sensing (ECIS) to assess intercellular adhesion in cells heterologously expressing β1, we find that inhibitory effects of βadp1 can persist for up to 5 hours. However, this acute inhibition is not sustained, with βadp1 effects on β1-mediated adhesion lost after 24 hours. We also determined that a short peptide (LQLEED) near the carboxyl-terminal portion of βadp1 inhibited adhesion in β1-expressing cells in a manner similar to βadp1. Paradoxically, dimeric peptides incorporating a repeat of the LQLEED sequence promoted intercellular adhesion at all time points studied over a 2-day time course. Inhibitory and agonistic peptides were found to effect β1 RIP, with βadp1increasing RIP continuously over 48 hours, whilst dimeric agonists acutely increased RIP at 6 hours post-treatment, but not thereafter. In the presence of DAPT, an inhibitor of RIP, the effects of βadp1 on ECIS-measured intercellular adhesion were lost, suggesting a relationship between RIP and inhibitory effects of the peptide. In sum, we identify novel SCN1B (β1/β1B) mimetic peptides with potential to inhibit and promote intercellular β1-mediated adhesion, possibly including by effects on β1 RIP, suggesting paths to development of anti-arrhythmic drugs targeting the perinexus.