1) To understand the properties of these channels using channel specific drugs like Tetradotoxin (TTX) and Tetraethylammonium (TEA).
2) To understand the role of selective blocking and complete blocking on action potential generation.
Tetradotoxin: An alkaloid neurotoxin, produced by certain species of puffer fish, tropical frogs, and salamanders, that selectively blocks voltage-sensitive Na+ channels; eliminates the initial Na+ current measured in voltage clamp experiments.
Tetraethlyammonium: A quaternary ammonium compound that selectively blocks voltage-sensitive K+ channels; eliminates the delayed K+ current measured in voltage clamp experiments.
Action potentials are known to be the source of communication transmitted from one part of the body to another through neurons, which are commonly represented as electrical signals. The main principle underlying this mechanism is regulated by ionic channels through selective permeability of ions with respect to the concentration gradient at a specific point of time, calculated by the amount of ions present outside and inside of the cell. From earlier exercise explanation (refer Modeling Action Potentials) it has become clear that depolarization occurs through fast Na+ channels and repolarization is through delayed rectifier K+ channels. With sufficient stimulus strength, the voltage of the neuronal membrane rapidly increases due to the influx of sodium ions thereby facilitating activation of feedback loop and decreases due to outflow of potassium ions. Flow of sodium ions into nerve cells is a necessary step in the conduction of nerve impulses in excitable nerve fibers and along axons. Understanding the properties of these ionic channels became a necessity to understand the underlying dynamics. Many postulates have been considered until mid-1960s which included permeation, binding and migration, passage through carriers, flow through pores, etc.
Around mid-1960s, Katz and Ricardo Miledi used Tetradotoxin (TTX) and Tetraethylammonium (TEA) in their attempt to study the properties of these channels. It is known that pharmacological experiments with molecules such as TTX and TEA, shown in Figure 1 provided the key to study channels as discrete entities (Kandel, 2000). Selective blocking of one of the channels enabled us to study the behavior and properties of the ionic channels as the action potentials result mainly due to the intricate interplay between sodium and potassium channels. Thereby, blocking one of the channels would enable us to study the properties of another channel.
Main principle of pharmacological class of drugs is that they are highly selective and they would bind to specific molecular components of specific regions of the neuron. Though many drugs and toxins exist to study these ion channels, we restrict our study to Tetradotoxin (TTX) and Tetraethlammonium (TEA) in this exercise as they are widely employed. Using these two agents (Tetradotoxin (TTX) and Tetraethlyammonium (TEA)), we can test our understanding of the ionic mechanisms of the action potential.
Figure 1: Left side of the above figure shows molecular structure of TTX and right side shows molecular structure of TEA.
Tetradotoxin (TTX), isolated from the Japanese puffer fish is a virulent poison known to cause respiratory paralysis, blocks conduction of nerve and muscle through its rather selective inhibition of the sodium-carrying mechanism. This drug should be administered at very low dose levels typically of the order 10-7 to 10-9 (micro-molecular concentrations) which acts in a reversible manner. It means that after the administration of the drug, the actual property of the channels can be reversed by washing with normal medium (early studies used sea water). The recovery can be partial or complete depending on the precision of the process applied. Experimental studies with voltage-clamp helped to show the suppression of the rise of sodium and potassium conductance normally occurring upon depolarization. TTX is known to block excitability through its selective inhibition of the sodium-carrying system without affecting the potassium-carrying system (Nakajima et al., 1962; Narahashi et al., 1960). From one of the studies conducted by Narahashi 1964a, this view is supported by the finding that maximum rate of rise of the action potential, which is indicative of the inward sodium current during activity is decreased much faster than is the rate of fall during the course of TTX block. This did not affect the resting potential. TTX much larger than the sodium ion, acts like a cork in a bottle, preventing the flow of sodium until it slowly diffuses off. TTX competes with the hydrated sodium cation and enters the Na+ channel where it binds. It is proposed that this binding results from the interaction of the positively charged guanidine group on the TTX and negatively charged carboxylate groups on side chains in the mouth of the channel.
Why this doesn’t have any effect on the host (Puffer fish)?
Sodium ion channel in the host must be different than that of the victim. The toxin might not have any effect, probably due to difference in the amino acid sequence of sodium channel. Protein of the sodium ion channel has undergone a mutation that changes the amino acid sequence making the channel insensitive to tetrodotoxin. The spontaneous mutation that causes this structural change is beneficial to the puffer fish because it allowed it to incorporate the symbiotic bacteria and utilize the toxin it produces to its best advantage. A single point mutation in the amino acid sequence of the sodium-ion channel in this species renders it immune from being bound and blockaded by TTX.
Tetraethylammonium(TEA) is synthesized from bromopentacarbonylrhenium by heating it with tetraethylammonium bromide in diglyme. It is a potassium-selective ion channel blocker. From the diagram you can observe that initial phase of the action potentials is identical, but note that it is much longer and does not have an after-hyperpolarization. There is a repolarization phase, but now the repolarization is due to the process of Na+ inactivation alone. There is no change in the resting potential. The channels in the membrane that endow the cell with the resting potential are different from the ones that are opened by voltage. They are not blocked by TEA. TEA only affects the voltage-dependent changes in K+ permeability. Perfusion of an axon by TEA also increases the duration of a propagated action potential but has no effect on its speed of propagation. It only affects only the fall time of the action potential. Bath application of TEA (1-10 mM) depolarized the resting potential, prolonged the action potential and increased the amplitude and duration of the ensuing passive depolarizing after-potential (DAP) in a dose-dependent and reversible manner. TEA increased the axonal input resistance and the slow time constant of the passive voltage response, not only in depolarized axons, but also in resting and hyperpolarized axons. TEA’s effects on the resting potential and action potential usually approached a steady state within 5 mins, whereas TEA’s effects on input resistance and on the amplitude and time course of the DAP increased progressively for 10-15 min or more, and persisted for 10-15 after removal of TEA from the bath.
TEA increases the duration of the action potential (Schmidt & Stampfli, 1966) by blocking depolarization-activated delayed rectifier K+ channels in the nodal axolemma. A motor nerve terminal stimulated in the presence of TEA releases more transmitter (Katz & Miledi, 1969; Benoit & Mambrini, 1970), and may discharge repetitive action potentials (Koketsu, 1958; Payton & Shand, 1966). TEA is also known to reverse the action of drugs such as tubocurarine, a non-depolarizing blocker. TEA evokes more release of the neurotransmitter and thus it will reverse the competitive antagonistic block of any drugs belonging to the curare family.
Along with these drugs, another drug by name Pronase is also studied with the help of neuron simulator which plays an important role in blocking sodium channel inactivation (acts as an antagonist to TTX). It’s been extensively used to analyze the kinetics of sodium channel activation.