Voltage-gated sodium channels
TTX is known to interfere with the function of VGSCs. VGSCs respond to the changes in membrane potential by opening and closing for sodium ions (Figure 1). At resting potential, the intracellular space is negatively charged relative to the extracellular space. When the membrane is depolarized to a threshold value, VGSCs open and the rapid influx of sodium ions further depolarizes the membrane. This increase in intracellular potential activates proximal VGSCs, resulting in a chain of channels opening sequentially – an action potential. The VGSC closes automatically, typically a few milliseconds after opening.
The selectivity filter and DEKA ring
TTX interacts with a “selectivity filter” inside the VGSC pore. The filter contains four amino acids: Asp on domain I, Glu on domain II, Lys on domain III, and Ala on domain IV (nicknamed DEKA). The four domains of a VGSC are homologous (Figure 2). In one study, fourteen different residue modifications were made to the DEKA ring and the IC50 of TTX increased in every case. Each amino acid of DEKA is necessary for optimal TTX binding, even Ala.
A model of the DEKA ring was proposed in 2000 (Figure 2). Lys forms a strong electrostatic interaction with Glu and a water bridge exists between Lys and Asp. A sodium ion has enough free energy to displace Lys and permeate the channel while a potassium ion (weaker Lewis acid) cannot. The guanidinium ion of TTX can also displace Lys and act as a bridge between Lys and Asp. The rest of TTX is too bulky to enter the pore however, so it blocks the channel.
Though the primary mechanism is TTX’s interaction with the DEKA ring, interactions between other parts of the molecule with surrounding residues stabilize TTX inside the channel. For example, the C11 hydroxyl of TTX is believed to form a hydrogen bond with Asp1532 of domain IV. Mutating the Asp to Asn doesn’t change TTX’s affinity while mutating it to Lys or Ala lowers it. Asp and Asn are similar in size so Asn can maintain the hydrogen bond while Lys is longer and Ala is shorter, so a hydrogen bond cannot form (Figure 3).
Furthermore, there is a cation-π interaction between TTX and P401/Y401 of the outer vestibule of domain I. Successive fluorination of the residue at position 401 decreases TTX affinity, suggesting a cation-π interaction is taking place. The π-system of an aromatic residue is weakened by fluorination because fluorine atoms withdraw electrons from the ring, which explains the pattern seen. A hydrophobic interaction is ruled out because mutating the 401 residue to Leu also decreases TTX affinity.