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PhunnyPhysiology
Neuronal Action Potential -
Pharmacological Inhibition of Na+ and K+ Channels
From the description of the action potential, it is clear that the changes that take place in the membrane potential result from ionic movements across the cell plasma membrane. These movements are made possible by the opening of voltage-gated Na+ channels (specific for Na+ ions) and voltage-gated K+ channels (specific for K+ ions). Movement of Na+ is into the cell down its electrochemical gradient, and this Na+ entry into the cell depolarizes the membrane and moves Vm close to VNa. Movement of K+ is out of the cell down its electrochemical gradient, and this outward K+ flow repolarizes, and even hyperpolarizes the membrane. Since movement of charge leads to an electrical current, these events can also be studied by using electrophysiological methods (Fig. 1). In physiological solutions, current is carried by ions such as Na+ and K+. We can carefully study the ionic currents passing through the membrane during the action potential by using drugs that specifically block either the voltage-gated Na+ channels or the voltage-gated K+ channels. Indeed, inhibition of Na+ and/or K+ channels by using specific pharmacological agents, has provided very clear information about the role these channels play in the neuronal action potential.
Tetrodotoxin (TTX) is a naturally-found poison that inhibits the voltage-gated Na+ channels. It is found in the liver and sex organs of marine puffer fish and other species of the order Tetraodontiformes (which includes porcupinefish, ocean sunfish, and triggerfish). Tetrodotoxin has also been found in other animals including the blue-ringed octopus and rough-skinned newt. It is interesting to note that TTX is actually produced by symbiotic bacteria living within the above-mentioned animals. Interestingly, puffer fish is a delicacy in some countries and, therefore, the ovaries must be carefully and completely removed before puffer fish is served. If done incorrectly, even minute quantities of ingested TTX can be fatal! Because of its ability to block action potentials and, hence, interfere with, or severely disrupt, the function of the nervous system, TTX is also considered to be a neurotoxin. Indeed, TTX can bind to voltage-gated Na+ channels at very low concentrations (nanomolar range). Poisoning at low levels, such as when ingesting improperly prepared puffer fish, can lead to numbness and/or tingling of the tongue. Moderate levels of TTX can lead to disturbances of the heart rhythm. Poisoning at high concentrations can paralyze the diaphragm leading to asphyxia and death.
Local anesthetics such as lidocaine (Xylocaine®) and procaine (Novacaine®) prevent the generation of action potentials by inhibiting voltage-gated Na+ channels of sensory neurons. Thus, depolarization elicited by sensory stimulation (generator or receptor potentials) does not lead to the generation of action potentials that can travel to the central nervous system. Lidocaine and procaine are commonly referred to as nerve blocking agents and, as we have mentioned, the molecular basis of their action is inhibition of the voltage-gated Na+ channels.
Similarly, voltage-gated K+ channels can be inhibited by specific agents. Tetraethyl ammonium (TEA), a quaternary ammonium cation, is one such agent that inhibits the voltage-gated K+ channels of neurons. As a blocker of voltage-gated K+ channels, TEA is very useful in elucidating the role K+ channels play in the neuronal action potential (see Fig. 1).
Pharmacological inhibition of Na+ and K+ channels.
Figure 1. Pharmacological inhibition of Na+ and K+ channels.
The neuronal action potential is characterized by rapid depolarization of the neuronal plasma membrane, which is then followed by repolarization, and even hyperpolarization of the membrane before the membrane potential returns to the resting value. The activity of voltage-gated Na+ and K+ channels is responsible for the characteristic features of the neuronal action potential. (A) Under voltage-clamp conditions, the currents passing through voltage-gated Na+ and K+ channels can be recorded. Here, using voltage-clamp, the membrane potential was rapidly changed from its resting level (&minus65 mV) to a value positive enough to ensure full opening (activation) of voltage-gated Na+ and K+ channels (upper red trace). (B) In response to the depolarizing voltage jump (red trace in panel A), the current passing through the membrane was recorded. The initial transient downward deflection (inward current) of the current trace corresponds to Na+ efflux through voltage-gated Na+ channels. The later delayed upward deflection (outward current) of the current trace corresponds to K+ efflux through voltage-gated K+ channels. (C) Tetrodotoxin (TTX), a blocker of voltage-gated Na+ channels, abolishes the inward current. This suggests that the inward current is due to the entry of Na+ through plasma membrane voltage-gated Na+ channels. Thus, the remaining current after TTX treatment is the delayed outward K+ current. (D) Tetraethyl ammonium (TEA), a blocker of voltage-gated K+ channels, abolishes the outward current. This suggests that the outward current is due to the efflux of K+ through plasma membrane voltage-gated K+ channels. Thus, the remaining current after TEA treatment is the transient inward Na+ current. Note: The traces shown are based on simulations with a classical Hodgkin-Huxley model of the squid giant axon. For simplicity, the capacitive and leak membrane currents were omitted.
Experimentally, TTX and TEA are very useful in investigating the role of Na+ and K+ channels in action potentials (Fig. 1). This can be demonstrated by showing the results of a voltage-clamp electrophysiology experiment. Recall that electrophysiology refers to the study of physiological phenomena by using electrical recordings. While several different electrophysiological approaches are used when studying biological phenomena, two methods are quite prevalent when studying the function of neurons: current-clamp and voltage-clamp. The method name describes the experimental manipulation. "Clamp" refers to keeping a parameter constant. In current clamp, the current passing across the membrane is controlled experimentally, and the membrane voltage changes that take place are measured. In fact, the voltage changes that were shown during the action potential are typically obtained by using current-clamp recordings. In voltage clamp, the membrane voltage is controlled experimentally, and the current passing across the membrane is measured. Special electronic feedback is used to maintain the membrane potential at a desired voltage, while measuring the current that passes through the membrane.
The results of a typical voltage-clamp experiment are shown in Fig. 1. Figure 1A shows the changes that take place in the membrane potential during the neuronal action potential. As mentioned above, the recorded changes in the membrane potential are obtained under current clamp conditions. We can also use voltage-clamp conditions to examine the ionic currents that pass through the neuronal plasma membrane during an action potential (Fig. 1). In a typical voltage-clamp experiment, the membrane potential is rapidly changed (by the investigator) from the resting value (around −70 mV) to a fixed value positive enough to ensure full opening (activation) of voltage-gated Na+ and K+ channels (Fig. 1A). This experimental maneuver simulates the physiological condition of depolarization, however, allows a reliable study of the function of voltage-gated ion channels at a fixed membrane voltage. The voltage jumps can be repeated at different membrane potentials in order to understand the voltage-dependence of ion channels. Voltage-clamp experiments show that there is a transient downward deflection (in the negative direction) of the current trace during the spike phase of the action potential, and there is a delayed upward deflection (in the positive direction) of the current trace during the repolarization and hyperpolarization phases of the action potential (Fig. 1B). A negative or downward deflection of the current trace is typically referred to as an inward current. A positive or upward deflection of the current trace is typically referred to as an outward current. Based on electrophysiological conventions, we know that a negative current value (i.e., inward current) can reflect either the movement of positive ions (cations) into the cell or negative ions (anions) out of the cell. A positive current value (i.e., outward current) can reflect either the movement of positive ions (cations) out of the cell or negative ions (anions) into the cell. Based on decades of experimentation, we know with certainty that the transient inward current observed during the action potential is carried by Na+ ions moving into the cell, and the delayed outward current is carried by K+ ions moving out of the cell.
Using the voltage-clamp experiments, a variety of experimental manipulations have been performed to understand the current that passes through the plasma membrane during the neuronal action potential. For example, the neuronal preparation can be studied in the absence of external Na+ in the physiological buffer that bathes the neuron. This can be done by replacing Na+ with the same concentration (to keep the buffer isosmolar) of another cation that does not permeate the pore of the voltage-gated Na+ channels (such as choline). In the absence of Na+ in the external medium, the inward current of the action potential is no longer observed suggesting that Na+ influx is responsible for this current.
Pharmacological tools have also been very helpful in elucidating the role of Na+ and K+ channels (Figs. 1C and 1D). Voltage-clamp current recordings of the action potential demonstrate that TTX (Na+ channel blocker) abolishes the inward Na+ current, and TEA (K+ channel blocker) abolishes the outward K+ current (Figs. 1C and 1D).






Posted: Thursday, July 5, 2012
Last updated: Friday, January 17, 2014