History: relevant studies
In 1964, Dr. Narahashi of Duke University elucidated the function of TTX with a breakthrough study. At that point, all that was known about the toxin was that it interferes with action potentials, in a manner similar to cocaine. Both TTX and cocaine seemed to stifle action potentials but did not affect resting potentials. He isolated the giant axons from a lobster species, Homarus americanus, clamped them at specific membrane potentials, and then stimulated them. Under normal conditions, the action potentials consisted of a sudden inward current (influx of sodium ions) followed by a gradual outward current (efflux of potassium ions). In the presence of TTX, the inward current was greatly diminished while the outward current was unaffected. This study revealed that TTX selectively inhibited the sodium channels and opened the door to further studies involving the toxin’s function.
Another study in 1974 showed that TTX interacts at a metal-coordinating site on the channel. The binding capacity of TTX was measured in the presence of different cations. The cations La3+, Ca2+, Be2+, Tl+, Li+ all reversibly inhibited TTX, showing that TTX’s binding site could bind monovalent, divalent, and trivalent cations. The apparent Kd’s of the cations were relatively high however, with some on the molar range. This agrees with the function of VGSCs though; strong binding is not desired if sodium ions are to be rapidly funneled through the channel. This study suggested the existence of negatively charged residues at the binding site of TTX.
Influence of sodium ions inside the channel pore
While attempting to create a model of TTX docked to a VGSC, researchers discovered an interesting phenomenon. In the study, a molecular dynamics simulation with predefined parameters for interaction strength and range was run. TTX was placed with the guanidinium group facing the selectivity filter and the simulation was started. The molecule was observed to exit the pore, orient itself perpendicular to the channel, and then re-enter, in a span of 20 ns. In the final position, TTX formed several interactions with the selectivity filter of the pore. It was also observed that TTX forms six interactions when a sodium ion is in the pore, and four interactions without it. Though sodium doesn’t directly interact with TTX, it likely induces conformational changes in the pore that stabilize TTX binding. This observation is in accordance with another study that showed TTX binding is stronger after membrane depolarization. Repetitive depolarization traps sodium ions inside the pore, which was shown to increase TTX affinity.
Hundreds of drugs have similar properties to TTX. Over 400 drugs have been screened for VGSC blocking activity and approximately 25% of them were positive. Saxitoxin (STX) and neosaxitoxin (NSTX) in particular, are compounds with structures and functions similar to TTX (Figure 1). They are both found in shellfish and are classified as paralytics shellfish poisoning (PSP) toxins. Both PSP toxins bind and inhibit VGSCs and induce many of the same symptoms as TTX. Both STX and NSTX feature two guandinium groups, with the only difference between them being a hydroxyl. While NSTX is relatively unknown, a substantial amount of research has been done on STX. One study looked at TTX and STX binding affinities when certain channel residues were mutated. Mutations to DEKA yielded similar changes in affinity for TTX and STX, indicating that they interact with DEKA in a similar manner. Some mutations to charged residues on the outer pore had a greater effect on TTX while other mutations had a greater effect on STX. Because STX has twice the charge as TTX, STX affinity was expected to change twice as much when charged residues were mutated. This was not observed however, confirming that more than just charge-interactions are at play. Based on the interactions studied, models of TTX and STX docking to the selectivity filter were created (Figure 2).