mu-Conotoxins as modulators of electrical signaling in nerve and muscle: Molecular basis of sodium channel block and discrimination among closely related channels

Voltage-gated sodium (Nav) channels are key players in the generation of electrical signals in muscle and nerves. Dysfunction of these channels can cause hyperexcitability leading to various diseases including cardiac arrhythmias, myotonias, epilepsies and pain. With a single underlying cause, hyperexcitability, in a wide spectrum of diseases, the identification of Nav subtype-specific blockers is of therapeutic importance. The muconotoxins from marine cone snails block Nav channel conduction and decrease excitability. The main objective of this work was to analyze the interactions between two peptide iconotoxins, PIIIA and KIIIA, and their target Nav channels, in order to gain a better understanding of the molecular basis of the binding affinity and selective targeting of these peptides for the particular Nav channel molecular subtypes that are present in different tissues.;In summary, this thesis adds persuasive elements to our understanding of how specific iconotoxin residues interact with the different Na v channel subtypes which underlie electrical excitability in specific tissues. These discoveries will aid in the design and development of subtype-specific Nav channel blockers of potential therapeutic relevance.;In this work, we used a combination of single-channel bilayer and whole-cell patch clamp recordings, in addition to molecular dynamics simulations to identify specific toxin and channel amino acids, which affect toxin binding. Most importantly, we provide evidence that neuronal channel targeting by PIIIA and KIIIA results from the concerted interactions of several amino acids, including a histidine located in the C-terminal section of the peptide. These differences arise partly from the identity of channel residues located near the selectivity filter. For KIIIA, we have identified a significant toxin binding "microsite" near the outer ring charge in channel domain III. This site is an important determinant of the targeting of the toxin to inhibit human Nav1.7, a channel important in pain signalling. Neutral substitution of certain toxin charges (KIIIA-H12A and KIIIA-R14A), increases the toxin's selectivity towards hNav1.7. Understanding how drugs can target this unique hNav1.7 site will aid the development of subtype specific blockers against this channel to help elevate pain.