Tetrodotoxin Resistance: Natural Experiments to Understand Voltage-Gated Sodium Channel Structure and Function

Abstract

Here we report data from our long-term studies of naturally occurring tetrodotoxin (TTX) resistant voltage-gated sodium channels (VGSCs) in TTX-resistant metazoans. These results identify critical biophysical properties in individual members of the VGSC gene family as well as differences between members. Members of the VGSC gene family are expressed in different excitable tissues and encode proteins with different biophysical properties. Characterizing isoform-specific properties is critical for understanding functional differences between excitable cells because, in nerve and muscle cells, the compliment of VGSCs expressed controls cell excitability. TTX is a neurotoxin used by diverse taxa including octopuses, newts, and fishes. The toxin causes paralysis and death by binding to and blocking the pore of VGSCs. Amino acid substitutions in the channel pore alter TTX binding as well as change biophysical properties such as voltage-dependence of activation and slow inactivation. Multiple species have evolved resistance to TTX through pore substitutions in their VGSCs. We have used these natural experiments to identify shared substitutions in TTX-resistant VGSC orthologs (e.g. NaV1.4) and paralogs (e.g. NaV1.5 and NaV1.7) of multiple TTX-resistant animals. By characterizing the properties of channels encoded by synthesized snake NaV1.4 genes, our work identifies interactions between pore residues and demonstrates that multiple convergent amino acid substitutions are compensatory. Specific amino acid changes rescue channel function when they occur in combination with substitutions that dramatically increase TTX resistance. Additionally, our work confirms that TTX resistance evolved prior to ecological exposure in snake and salamander lineages. These patterns suggest that selective pressures on channel function rather than a requirement for TTX resistance supported alterations in channel structure in our study taxa. By comparing convergent and divergent substitutions, we identify critical biophysical differences between members of the VGSC gene family.