Characterization of Novel Site 3 Modulators to Explore Inactivation of the VGSC Isoform Nav1.7

Author

John William Johnson, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2025

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Tischkau, Shelley

Abstract

Pain is a condition that ultimately affects all people throughout the course of their lifetime. It is attributed to a wide variety of causes and becomes an all-encompassing focus for those who suffer disproportionally from neuropathic and chronic forms of pain. Some treatment options are available, but many fail to provide sufficient support to a variety of pain conditions. Opioids account for a large portion of pain management treatment with some success but have a variety of unwanted side effects and limited efficacy in certain cases. Additionally, opioid abuse continues to increase in our modern culture and leads to premature death of thousands in the United States. The voltage-gated sodium channel (VGSC) isoform Nav1.7 has emerged as a promising target for a novel class of analgesics that may lessen or replace the need for opioids. VGSCs are responsible for the initiation and propagation of action potentials in excitable cells and Nav1.7 are directly linked to nociception in the periphery. Venoms comprise a large library of diverse compounds, i.e. small molecules, peptides, and proteins, that nature has evolved to modulate ion channels potently and often selectively for purposes that vary depending on the species. Venom peptides that target Nav1.7 channels can be studied to highlight the structure-function relationship that underpins the protein-protein interactions (PPI) involved in their modulation. While there are three physical state changes that a single VGSC undergoes throughout an action potential, many venom compounds have been shown to affect inactivation when binding to site 3 of the Nav1.7 channels. To further understand this type of modulation, the venoms of Heteroctenus junceus and Poecilotheria regalis have been screened to search for novel peptides that are suspected to bind to site 3 of Nav1.7 and affect inactivation. Venoms were collected by electrostimulation from specimens under anesthesia. Lyophilized venom was then separated into fractions using size exclusion and reverse phase chromatography methods. By conventional, whole-cell patch clamping methods, venom fractions were tested on Nav1.7 channels, predominately expressed in catecholamine A differentiated (CAD) cells utilizing a variety of voltage protocols. Fractions containing peptides that displayed Nav1.7, site 3 modulatory effects were purified further using reverse phase chromatography. The detailed structure of these peptides was determined by de novo sequencing using UPLC-HRMS. In this study, the venoms of two arachnid venoms, Heteroctenus junceus and Poecilotheria regalis, were investigated to assess their effects on inactivation with two peptides, HtsTX-1 and PcaTX-2, being isolated from each respectively. These peptides were selected due to their similarity to previously described site 3 modulators, with both causing a significant delay in inactivation. They were also shown to enhance the recovery from steady-state fast inactivation. Closed-state inactivation (CSI) is a physiologically relevant mechanism that VGSC may undergo that allows channels to inactivate without first opening. The effect venom toxins have on CSI has not been heavily studied. Both HtsTX-1 and PcaTX-2 here are shown to decrease VGSC’s ability to undergo CSI. This begins to provide an understanding of how CSI can be modulated. This knowledge could assist in further studies to enhance CSI, which would place VGSC in a refractory position without being activated. Thus, enhancing CSI could be used to treat a variety of disease states that are caused by underlying neuronal hyperexcitability.