molecular mechanisms for action potential

Ionic Mechanisms of Action Potentials

Voltage-Dependent Conductances

Na⁺ is critical for the action potential in nerve cells. Action potentials are repeatedly initiated as the extracellular concentration of Na⁺ is modified. As the concentration of sodium in the extracellular solution is reduced, the action potentials become smaller.

The straight line is predicted by the Nernst equation (assuming the membrane was exclusively permeable to Na⁺). There is a good fit between the data and the values predicted by a membrane that is exclusively permeable to Na⁺. The experiment gives experimental support to the notion that at the peak of the action potential, the membrane becomes highly permeable to sodium.

However, there are some deviations between what is measured and what is predicted by the Nernst equation. Why? One reason for the deviation is the continued K⁺ permeability. If there is continued K⁺ permeability, the membrane potential will never reach its ideal value (the sodium equilibrium potential) because the diffusion of K⁺ ions tends to make the cell negative. This point can be understood with the aid of the GHK equation.

An action potential is bounded by a region bordered on one extreme by the K⁺ equilibrium potential (-75 mV) and on the other extreme by the Na⁺ equilibrium potential (+55 mV). The resting potential is -60 mV. Note that the resting potential is not equal to the K⁺ equilibrium potential because, as discussed previously, there is a small resting Na⁺ permeability that makes the cell slightly more positive than EK. In principle, any point along the trajectory of action potential can be obtained simply by varying alpha in the GHK equation. If alpha is very large, the Na⁺ terms dominate, and according to the GHK equation, the membrane potential will move towards the Na⁺ equilibrium potential. The peak of the action potentials approaches but does not quite reach ENa, because the membrane retains its permeability to K⁺.

How is it possible for a cell to initially have a resting potential of -60 mV and then, in response to some stimulus (a brief transient depolarization which reaches threshold), change in less than one millisecond to having a potential of approximately +40 mV? In the 1950's, Hodgkin and Huxley, two British neurobiologists, provided a hypothesis for this transition. They suggested that the properties of some Na⁺ channels in nerve cells (and muscle cells) were unique in that these channels were normally closed but could be opened by a depolarization. This simple hypothesis of voltage-dependent Na⁺ channels goes a long way toward explaining the initiation of the action potential. Suppose a small depolarization causes some of the Na⁺ channels to open. The key point is that the increase in Na⁺ permeability would produce a greater depolarization, which will lead to an even greater number of Na⁺ channels opening and the membrane potential becoming even more depolarized. Once some critical level is reached a positive feedback or regenerative cycle will be initiated, causing the membrane potential to depolarize rapidly from -60 mV to a value approaching the Na⁺ equilibrium potential.

In order to test the Na⁺ hypothesis for the initiation of the action potential, it is necessary to stabilize the membrane potential at a number of different levels and measure the permeability at those potentials. An electronic device known as a voltage-clamp amplifier can "clamp" or stabilize the membrane potential to any desired level and measure the resultant current required for that stabilization. The amount of current necessary to stabilize the potential is proportional to the permeability. Hodgkin and Huxley clamped the membrane potential to various levels and measured the changes in Na⁺ conductances (an electrical term for permeability, which for the present discussion can be used interchangeably). The more the cell is depolarized, the greater is the Na⁺ conductance. Thus, the experiment provided support for the existence of voltage-dependent Na⁺ channels.

2.2 Na⁺ Inactivation

observation also indicates an important property of the voltage-dependent Na⁺ channels. Note that the permeability increases rapidly and then, despite the fact that the membrane potential is clamped, the permeability decays back to its initial level. This phenomenon is called inactivation. The Na⁺ channels begin to close, even in the continued presence of the depolarization. Inactivation contributes to the repolarization of the action potential. However, inactivation is not enough by itself to account fully for the repolarization.

2.3 Voltage-Dependent K⁺ Conductance

In addition to voltage-dependent changes in Na⁺ permeability, there are voltage-dependent changes in K⁺ permeability. These changes can be measured with the voltage-clamp technique as well. The figure shown to above indicates the changes in K⁺ conductance as well as the Na⁺ conductance. There are two important points.

First, just as there are channels in the membrane that are permeable to Na⁺ that are normally closed but then open in response to a voltage, there are also channels in the membrane that are selectively permeable to K⁺. These K⁺ channels are normally closed, but open in response to depolarization.

Second, a major difference between the changes in the K⁺ channels and the changes in the Na⁺ channels is that the K⁺ channels are slower to activate or open. (Some K⁺ channels also do not inactivate.) Note that the return of the conductance at the end of the pulse is not the process of inactivation. With the removal of the pulse, the activated channels are deactivated.

2.4 Sequence of Conductance Changes Underlying the Nerve Action Potential

Some initial depolarization (e.g., a synaptic potential) will begin to open the Na⁺ channels. The increase in the Na⁺ influx leads to a further depolarization.

A positive feedback cycle rapidly moves the membrane potential toward its peak value, which is close but not equal to the Na⁺ equilibrium potential. Two processes which contribute to repolarization at the peak of the action potential are then engaged. First, the Na⁺ conductance starts to decline due to inactivation. As the Na⁺ conductance decreases, another feedback cycle is initiated, but this one is a downward cycle. Sodium conductance decreases, the membrane potential begins to repolarize, and the Na⁺ channels that are open and not yet inactivated are deactivated and close. Second, the K⁺ conductance increases. Initially, there is very little change in the K⁺ conductance because these channels are slow to open, but by the peak of the action potential, the K⁺ conductance begins to increase significantly and a second force contributes to repolarization. As the result of these two forces, the membrane potential rapidly returns to the resting potential. At the time it reaches -60 mV, the Na⁺ conductance has returned to its initial value. Nevertheless, the membrane potential becomes more negative (the undershoot or the hyperpolarizing afterpotential).

The key to understanding the hyperpolarizing afterpotential is in the slowness of the K⁺ channels. Just as the K⁺ channels are slow to open (activate), they are also slow to close (deactivate). Once the membrane potential starts to repolarize, the K⁺ channels begin to close because they sense the voltage. However, even though the membrane potential has returned to -60 mV, some of the voltage-dependent K⁺ channels remain open. Thus, the membrane potential will be more negative than it was initially. Eventually, these K⁺ channels close, and the membrane potential returns to -60 mV.

Why does the cell go through these elaborate mechanisms to generate an action potential with a short duration? Recall how information is coded in the nervous system. If the action potential was about one msec in duration, the frequency of action potentials could change from once a second to a thousand a second. Therefore, short action potentials provide the nerve cell with the potential for a large dynamic range of signaling.

2.5 Pharmacology of the Voltage-Dependent Membrane Channels

Some chemical agents can selectively block voltage-dependent membrane channels. Tetrodotoxin (TTX), which comes from the Japanese puffer fish, blocks the voltage-dependent changes in Na⁺ permeability, but has no effect on the voltage-dependent changes in K⁺ permeability. This observation indicates that the Na⁺ and K⁺ channels are unique; one of these can be selectively blocked and not affect the other. Another agent, tetraethylammonium (TEA), has no effect on the voltage-dependent changes in Na⁺ permeability, but it completely abolishes the voltage-dependent changes in K⁺ permeability.

Use these two agents (TTX and TEA) to test your understanding of the ionic mechanisms of the action potential. What effect would treating an axon with TTX have on an action potential? An action potential would not occur because an action potential in an axon cannot be initiated without voltage-dependent Na⁺ channels. How would TEA affect the action potential? It would be longer and would not have an undershoot.

In the presence of TEA the initial phase of the action potential 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. Note that in the presence of TEA, 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.

2.6 Pumps and Leaks

It is easy to receive the impression that there is a "gush" of Na⁺ that comes into the cell with each action potential. Although, there is some influx of Na⁺, it is minute compared to the intracellular concentration of Na⁺. The influx is insufficient to make any noticeable change in the intracellular concentration of Na⁺. Therefore, the Na⁺ equilibrium potential does not change during or after an action potential. For any individual action potential, the amount of Na⁺ that comes into the cell and the amount of K⁺ that leaves are insignificant and have no effect on the bulk concentrations. However, without some compensatory mechanism, over the long-term (many spikes), Na⁺ influx and K⁺ efflux would begin to alter the concentrations and the resultant Na⁺ and K⁺ equilibrium potentials. The Na⁺-K⁺ pumps in nerve cells provide for the long-term maintenance of these concentration gradients. They keep the intracellular concentrations of K⁺ high and the Na⁺ low, and thereby maintain the Na⁺ equilibrium potential and the K⁺ equilibrium potential. The pumps are necessary for the long-term maintenance of the "batteries" so that resting potentials and action potentials can be supported.

2.7 Types of Membrane Channels

So far, two basic classes of channels, voltage-dependent or voltage-gated channels and voltage-independent channels, have been considered. Voltage-dependent channels can be further divided based on their permeation properties into voltage-dependent Na⁺ channels and voltage-dependent K⁺ channels. There are also voltage-dependent Ca²⁺ channels (see chapter on Synaptic Transmission). Indeed, there are multiple types of Ca²⁺ channels and voltage-dependent K⁺ channels. Nevertheless, all these channels are conceptually similar. They are membrane channels that are normally closed and as a result of changes in potential, the channel (pore) is opened. The amino acid sequence of these channels is known in considerable detail and specific amino acid sequences have been related to specific aspects of channel function (e.g., ion selectivity, voltage gating, inactivation). A third major channel class, the transmitter-gated or ligand-gated channels, will be described later.

2.8 Channelopathies

Ion channel mutations have been identified as a possible cause of a wide variety of inherited disorders. Several disorders involving muscle membrane excitability have been associated with mutations in calcium, sodium and chloride channels as well as acetylcholine receptors and have been labeled ‘channelopathies’. It is possible that movement disorders, epilepsy and headache, as well as other rare inherited diseases, might be linked to ion channels. The manifestations and mechanisms of channelopathies affecting neurons are reviewed in Kullman, 2002. The existence of channelopathies may provide insights into the variety of cellular mechanisms associated with the misfunctioning of neuronal circuits.

2.9 Absolute and Relative Refractory Periods

The absolute refractory period is a period of time after the initiation of one action potential when it is impossible to initiate a second action potential no matter how much the cell is depolarized. The relative refractory period is a period after one action potential is initiated when it is possible to initiate a second action potential, but only with a greater depolarization than was necessary to initiate the first. The relative refractory period can be understood at least in part by the hyperpolarizing afterpotential. Assume that an initial stimulus depolarized a cell from -60 mV to -45 mV in order to reach threshold and then consider delivering the same 15-mV stimulus sometime during the after-hyperpolarization. The stimulus would again depolarize the cell but the depolarization would be below threshold and insufficient to trigger an action potential. If the stimulus was made larger, however, such that it again was capable of depolarizing the cell to threshold (-45 mV), an action potential could be initiated.

The absolute refractory period can be explained by the dynamics of the process of Na⁺-inactivation. Here, two voltage clamp pulses are delivered. The first pulse produces a voltage-dependent increase in the Na⁺ permeability which then undergoes the process of inactivation. If the two pulses are separated sufficiently in time, the second pulse produces a change in the Na⁺ conductance, which is identical to the first pulse. However, if the second pulse comes soon after the first pulse, then the change in Na⁺ conductance produced by the second pulse is less than that produced by the first. Indeed, if the second pulse occurs immediately after the first pulse, the second pulse produces no change in the Na⁺ conductance. Therefore, when the Na⁺ channels open and spontaneously inactivate, it takes time (several msec) for them to recover from that inactivation. This process of recovery from inactivation underlies the absolute refractory period. During an action potential the Na⁺ channels open and then they become inactivated. Therefore, if a second stimulus is delivered soon after the one that initiated the first spike, there will be few Na⁺ channels available to be opened by the second stimulus because they have been inactivated by the first action potential.