Toxins, Targets, and Time: The Evolutionary Biochemistry of Sodium Channels and Tetrodotoxin Resistance

Abstract

Understanding how protein sequence variation translates into functional and adaptive phenotypes remains a central challenge in evolutionary biology. This dissertation integrates phylogenetic reconstruction, molecular docking, and molecular dynamics simulations to investigate the evolution of tetrodotoxin (TTX) resistance in voltage-gated sodium (NaV) channels. TTX is a potent neurotoxin that binds to the pore of NaV channels, blocking sodium influx, thereby preventing action potentials, leading to paralysis and death. Many organisms possess TTX as a defense against predation, pressuring predators to evolve resistance. Resistance to TTX has evolved repeatedly across diverse taxa, providing a powerful system for examining the interplay between ecological interactions, molecular evolution, and protein structure and dynamics. However, key questions regarding how sequences, chemistry, and molecular dynamics result in phenotypic differences that have consequences for ecological relationships remain. First, I reconstruct the evolutionary history of two distinct sodium channels, NaV1 and NaV2, across deuterostomes, with a focus on pore loop regions that mediate ion selectivity and TTX binding. Ancestral sequence reconstruction reveals that NaV2 channels possess a conserved residues associated with high TTX resistance, suggesting that an inherence resistance-associated state predated the diversification of many modern lineages. In contrast, NaV1 channels evolved from a more TTX-sensitive background an exhibit repeated, lineage specific substitutions that converge on similar functional outcomes. Second, I use molecular dynamics simulations to characterize how TTX binding and resistance-associated mutations alter channel dynamics, revealing differences in the spatial distribution and magnitude of dynamical changes between sensitive and resistance channels. Third, I examine the interactions between NaV channels and structurally diverse TTX analogs using molecular docking, revealing how toxin chemistry plays a role in the evolution of a predator and prey arms race. Together, these results demonstrate that the evolution of TTX resistance is shaped by historical contingency, structural constraints, and chemical diversity. By linking sequence evolution to structural dynamics and toxin interactions, this work provides a framework for understanding how adaptive phenotypes emerge via molecular variation.