Propagated Signaling: The Action Potential

The Action Potential Is Generated by the Flow of Ions Through Voltage-Gated Channels

Sodium and Potassium Currents Through Voltage-Gated Channels Are Recorded with the Voltage Clamp

Voltage-Gated Sodium and Potassium Conductances Are Calculated from Their Currents

The Action Potential Can Be Reconstructed from the Properties of Sodium and Potassium Channels

Variations in the Properties of Voltage-Gated Ion Channels Expand the Signaling Capabilities of Neurons

The Nervous System Expresses a Rich Variety of Voltage-Gated Ion Channels

Gating of Voltage-Sensitive Ion Channels Can Be Influenced by Various Cytoplasmic Factors

Excitability Properties Vary Between Regions of the Neuron

Excitability Properties Vary Between Types of Neurons

The Mechanisms of Voltage-Gating and Ion Permeation Have Been Inferred from Electrophysiological Measurements

Voltage-Gated Sodium Channels Open and Close in Response to Redistribution of Charges Within the Channel

Voltage-Gated Sodium Channels Select for Sodium on the Basis of Size, Charge, and Energy of Hydration of the Ion

Voltage-Gated Potassium, Sodium, and Calcium Channels Stem from a Common Ancestor and Have Similar Structures

X-Ray Crystallographic Analysis of Voltage-Gated Channel Structures Provides Insight into Voltage-Gating

The Diversity of Voltage-Gated Channel Types Is Generated by Several Genetic Mechanisms

An Overall View

NERVE CELLS ARE ABLE TO CARRY electrical signals over long distances because the long-distance signal, the action potential, is continually regenerated and thus does not attenuate as it moves down the axon. In Chapter 6 we saw how an action potential arises from sequential changes in the membrane’s permeability to Na⁺, and K⁺ ions. We also considered how the membrane’s passive properties influence the speed at which action potentials are conducted. In this chapter we focus on the voltage-gated ion channels that are critical for generating and propagating action potentials and consider how these channels are responsible for many important features of a neuron’s electrical excitability.

Action potentials have four properties important for neuronal signaling. First, they have a threshold for initiation. As we saw in Chapter 6, in many nerve cells the membrane behaves as a simple resistor in response to small hyperpolarizing or depolarizing current steps. The membrane voltage changes in a graded manner as a function of the size of the current step according to Ohm’s law, (in terms of conductance, ). However, as the size of the depolarizing current increases, eventually a threshold voltage is reached, typically at around –50 mV, at which point an action potential is generated (see Figure 6-2C). Second, the action potential is an all-or-none event. The size and shape of an action potential initiated by a large depolarizing current is the same as that of an action potential evoked by a current that just surpasses the threshold.¹ Third, the action potential is conducted without decrement. It has a self-regenerative feature that keeps the amplitude constant, even when it is conducted over great distances. Fourth, the action potential is followed by a refractory period. For a brief time after an action potential is generated, the neuron’s ability to fire a second action potential is suppressed. The refractory period limits the frequency at which a nerve can fire action potentials, and thus limits the information-carrying capacity of the axon.

These four properties of the action potential—initiation threshold, all-or-none nature, conduction without decrement, and refractory period—are unusual for biological processes, which typically respond in a graded fashion to changes in the environment. Biologists were puzzled by these properties for almost 100 years after the action potential was first measured in the mid 1800s. Finally, in the late 1940s and early 1950s studies of the membrane properties of the giant axon of the squid by Alan Hodgkin, Andrew Huxley, and Bernard Katz provided the first quantitative insight into the mechanisms underlying the action potential.

The Action Potential Is Generated by the Flow of Ions Through Voltage-Gated Channels

An important early insight into how action potentials are generated came from an experiment performed by Kenneth Cole and Howard Curtis. While recording from the giant axon of the squid they found that ion conductance across the membrane increases dramatically during the action potential (Figure 7-1). This discovery provided the first evidence that the action potential results from changes in the flux of ions through channels in the membrane. It also raised two central questions: Which ions are responsible for the action potential, and how is the conductance of the membrane regulated?

Figure 7-1 The action potential results from an increase in ionic conductance of the axon membrane. This historic recording from an experiment conducted in 1939 by Kenneth Cole and Howard Curtis shows the oscilloscope record of an action potential superimposed on a simultaneous record of membrane conductance.

A key insight into this problem was provided by Alan Hodgkin and Bernard Katz, who found that the amplitude of the action potential is reduced when the external Na⁺ concentration is lowered, indicating that Na⁺ influx is responsible for the rising phase of the action potential. They proposed that depolarization of the cell above threshold causes a brief increase in the cell membrane’s permeability to Na⁺, during which Na⁺ permeability overwhelms the dominant K⁺ permeability of the resting cell membrane, thereby driving the membrane potential towards E_Na. Their data also suggested that the falling phase of the action potential was caused by a later increase in K⁺ permeability.

Sodium and Potassium Currents Through Voltage-Gated Channels Are Recorded with the Voltage Clamp

This insight of Hodgkin and Katz raised a further question. What mechanism is responsible for regulating the changes in the Na⁺, and K⁺ permeabilities of the membrane? Hodgkin and Andrew Huxley reasoned that the Na⁺, and K⁺ permeabilities were regulated directly by the membrane voltage. To test this hypothesis they systematically varied the membrane potential in the squid giant axon and measured the resulting changes in the conductance of voltage-gated Na⁺, and K⁺ channels. To do this they made use of a new apparatus, the voltage clamp, developed by Kenneth Cole.

Prior to the availability of the voltage-clamp technique, attempts to measure Na⁺, and K⁺ conductance as a function of membrane potential had been limited by the strong interdependence of the membrane potential and the gating of Na⁺, and K⁺ channels. For example, if the membrane is depolarized sufficiently to open some of the voltage-gated Na⁺ channels, the influx of Na⁺ through these channels causes further depolarization. The additional depolarization causes still more Na⁺ channels to open and consequently induces more inward Na⁺ current:

This positive feedback cycle, which eventually drives the membrane potential to the peak of the action potential, makes it impossible to achieve a stable membrane potential. A similar coupling between current and membrane potential complicates the study of the voltage-gated K⁺ channels.

The voltage clamp interrupts the interaction between the membrane potential and the opening and closing of voltage-gated ion channels. It does so by adding or withdrawing a current from the axon that is equal to the current flowing through the voltage-gated membrane channels. In this way the voltage clamp prevents the membrane potential from changing. The amount of current that must be generated by the voltage clamp to keep the membrane potential constant provides a direct measure of the current flowing across the membrane (Box 7-1). Using the voltage-clamp technique, Hodgkin and Huxley provided the first complete description of the ionic mechanisms underlying the action potential.

One advantage of the voltage clamp is that it readily allows the ionic and capacitive components of membrane current to be analyzed separately. As described in Chapter 6, the membrane potential V_m is proportional to the charge Q_m on the membrane capacitance C_m. When V_m is not changing, Q_m is constant, and no capacitive current (ΔQ_m/Δt) flows. Capacitive current flows only when V_m is changing. Therefore when the membrane potential changes in response to a very rapid step of command potential, capacitive current flows only at the beginning and end of the step. Because the capacitive current is essentially instantaneous, the ionic currents that subsequently flow through the voltage-gated channels can be analyzed separately.

Measurements of these ionic currents can be used to calculate the voltage and time dependence of changes in membrane conductance caused by the opening and closing of Na⁺, and K⁺ channels. This information provides insights into the properties of these two types of channels.

A typical voltage-clamp experiment starts with the membrane potential clamped at its resting value. When a 10 mV depolarizing step is applied, a very brief outward current instantaneously discharges the membrane capacitance by the amount required for a 10 mV depolarization. This capacitive current (I_c) is followed by a smaller outward current that persists for the duration of the voltage step. This steady ionic current flows through the nongated resting ion channels of the membrane, which we refer to here as leakage channels (see Box 6-2). The current through these channels is called the leakage current, I_l. The total conductance of this population of channels is called the leakage conductance (g_l). At the end of the step a brief inward capacitive current repolarizes the membrane to its initial voltage and the total membrane current returns to zero (Figure 7-3A).

Figure 7-3 A voltage-clamp experiment demonstrates the sequential activation of two types of voltage-gated channels.

A. A small depolarization (10 mV) elicits capacitive and leakage currents (I_c and I_l, respectively), the components of the total membrane current (I_m).

B. A larger depolarization (60 mV) results in larger capacitive and leakage currents, plus a time-dependent inward ionic current followed by a time-dependent outward ionic current.

Top: Total (net) current in response to the depolarization. Middle: Individual Na⁺, and K⁺ currents. Depolarizing the cell in the presence of tetrodotoxin (TTX), which blocks the Na⁺ current, or in the presence of tetraethylammonium (TEA), which blocks the K⁺ current, reveals the pure K⁺, and Na⁺ currents (I_K and I_Na, respectively) after subtracting I_c and I_l. Bottom: Voltage step.

If a large depolarizing step is commanded, the current record is more complicated. The capacitive and leakage currents both increase in amplitude. In addition, shortly after the end of the capacitive current and the start of the leakage current, an inward (negative) current develops; it reaches a peak within a few milliseconds, declines, and gives way to an outward current. This outward current reaches a plateau that is maintained for the duration of the voltage step (Figure 7-3B).

A simple interpretation of these results is that the depolarizing voltage step sequentially turns on two types of voltage-gated channels that select for two distinct ions. One type of channel conducts ions that generate an inward current, while the other conducts ions that generate an outward current. Because these two oppositely directed currents partially overlap in time, the most difficult task in analyzing voltage-clamp experiments is to determine their separate time courses.

Hodgkin and Huxley achieved this separation by changing ions in the bathing solution. By replacing Na⁺ with a larger, impermeant cation (choline · H⁺), they eliminated the inward Na⁺ current. Later, the task of separating inward and outward currents was made easier by the discovery of drugs or toxins that selectively block the different classes of voltage-gated channels (Figure 7-4). Tetrodotoxin, a poison from a certain Pacific puffer fish, blocks the voltage-gated Na⁺ channel with a very high potency in the nanomolar range of concentration. (Ingestion of only a few milligrams of tetrodotoxin from improperly prepared puffer fish, consumed as the Japanese sushi delicacy fugu, can be fatal.) The cation tetraethylammonium specifically blocks the squid axon voltage-gated K⁺ channel.

Figure 7-4 Drugs that block voltage-gated Na⁺, and K⁺ channels.

A. Tetrodotoxin and saxitoxin both bind to Na⁺ channels with a very high affinity. Tetrodotoxin is produced by certain puffer fish, newts, and frogs. Saxitoxin is synthesized by the dinoflagellates Gonyaulax, which are responsible for red tides. Consumption of clams or other shellfish that have fed on the dinoflagellates during a red tide causes paralytic shellfish poisoning.

B. Tetraethylammonium is a cation that blocks certain voltage-gated K⁺ channels with a relatively low affinity. The plus signs represent positive charge. 4-Aminopyridine is another blocker of K⁺ channels. It is used to improve conduction of nerve impulses in patients with multiple sclerosis.

When tetraethylammonium is applied to the axon to block the K⁺ channels, the total membrane current (I_m) consists of I_c, I_l, and I_Na. The leakage conductance, g_l, is constant; it does not vary with V_m or with time. Therefore the leakage current I_l can be readily calculated and subtracted from I_m, leaving I_Na and I_c. Because I_c occurs only briefly at the beginning and end of the pulse, it is easily isolated by visual inspection, revealing the pure I_Na. Similarly, I_K can be measured when the Na⁺ channels are blocked by tetrodotoxin (Figure 7-3B).

By stepping the membrane to a wide range of potentials, Hodgkin and Huxley were able to measure the Na⁺, and K⁺ currents over the entire voltage extent of the action potential (Figure 7-5). They found that the Na⁺, and K⁺ currents vary as a graded function of the membrane potential. As the membrane voltage is made more positive, the outward K⁺ current becomes larger. The inward Na⁺ current also becomes larger with increases in depolarization, up to a certain extent. However, as the voltage becomes more and more positive, the Na⁺ current eventually declines in amplitude. When the membrane potential is +55 mV, the Na⁺ current is zero. Positive to +55 mV, the Na⁺ current reverses direction and becomes outward.

Figure 7-5 The magnitude and polarity of the Na⁺, and K⁺ membrane currents vary with the amplitude of membrane depolarization. Left: With progressive depolarization the voltage-clamped membrane K⁺ current increases monotonically, because both g_K and (V_mE_K), the driving force for K⁺, increase with increasing depolarization. The voltage during the depolarization is indicated at left. The direction and magnitude of the chemical (E_K) and electrical driving force on K⁺, as well as the net driving force, is given by arrows at the right of each trace. (Up arrows = outward force; down arrows = inward force.)

Right: At first the Na⁺ current becomes increasingly inward with increasing depolarization due to the increase in g_Na. However, as the membrane potential approaches E_Na (+55 mV) the magnitude of the inward Na⁺ current begins to decrease due to the decrease in inward driving force (V_mE_Na). Eventually, I_Na goes to zero when the membrane potential reaches E_Na. At depolarizations positive to E_Na, the sign of (V_m –E_Na) reverses, and I_Na becomes outward. Arrows at the right of each trace show chemical (E_Na) and electrical driving forces and net Na⁺ driving force.

Hodgkin and Huxley explained this behavior by a very simple model in which the size of the Na⁺, and K⁺ currents is determined by two factors. The first is the magnitude of the Na⁺ or K⁺ conductance, g_Na or g_K, which reflects the number of Na⁺ or K⁺ channels open at any instant (see Chapter 6). The second factor is the electrochemical driving force on Na⁺ ions (V_mE_Na) or K⁺ ions (V_mE_K). The model is thus expressed as:

According to this model the amplitudes of I_Na and I_K change as the voltage is made more positive because there is an increase in g_Na and g_K. The Na⁺, and K⁺ conductances increase because the opening of the Na⁺, and K⁺ channels is voltage-dependent. The currents also change in response to changes in the electrochemical driving forces.

Both I_Na and I_K initially increase in amplitude as the membrane is made more positive because g_Na and g_K increase steeply with voltage. However, as the membrane potential approaches E_Na (+55 mV), I_Na declines because of the decrease in inward driving force, even though g_Na is large. That is, the positive membrane voltage now opposes the influx of Na⁺ down its chemical concentration gradient. At +55 mV the chemical and electrical driving forces are in balance so there is no net I_Na, even though g_Na is quite large. As the membrane is made positive to E_Na, the driving force on Na⁺ becomes positive. That is, the electrical driving force pushing Na⁺ out is now greater than the chemical driving force pulling Na⁺ in, and hence I_Na becomes outward. The behavior of I_K is simpler; because E_K is quite negative (–75 mV), both g_K and the outward driving force on K⁺ become larger as the membrane is made more positive, thereby increasing the outward K⁺ current.

Voltage-Gated Sodium and Potassium Conductances Are Calculated from Their Currents

From the two preceding equations Hodgkin and Huxley were able to calculate g_Na and g_K by dividing measured Na⁺, and K⁺ currents by the known Na⁺, and K⁺ electrochemical driving forces. Their results provide direct insight into how membrane voltage controls channel opening because the values of g_Na and g_K reflect the number of open Na⁺, and K⁺ channels (Box 7-2).

Measurements of g_Na and g_K at various levels of membrane potential reveal two functional similarities and two differences between the Na⁺, and K⁺ channels. Both types of channels open in response to depolarization. Also, as the size of the depolarization increases, the probability and rate of opening increase for both types of channels. The Na⁺, and K⁺ channels differ, however, in the rate at which they open and in their responses to prolonged depolarization. At all levels of depolarization the Na⁺ channels open more rapidly than K⁺ channels (Figure 7-7). When the depolarization is maintained for some time, the Na⁺ channels begin to close, leading to a decrease of inward current. The process by which Na⁺ channels close during a prolonged depolarization is termed inactivation.

Thus depolarization causes Na⁺ channels to switch between three different states—resting, activated, or inactivated—which represent three different conformations of the Na⁺ channel protein (see Figure 5-7). In contrast, squid axon K⁺ channels do not inactivate; they remain open as long as the membrane is depolarized, at least for voltage-clamp depolarizations lasting up to tens of milliseconds (Figure 7-7).

Figure 7-7 The responses of K⁺, and Na⁺ channels to prolonged depolarization. Increasing depolarizations elicit graded increases in K⁺, and Na⁺ conductance (g_Na and g_K), which reflect the proportional opening of thousands of voltage-gated K⁺, and Na⁺ channels. The Na⁺ channels open more rapidly than the K⁺ channels. During a maintained depolarization the Na⁺ channels close after opening because of the closure of an inactivation gate. The K⁺ channels remain open because they lack a fast inactivation process.

In the inactivated state the Na⁺ channel cannot be opened by further membrane depolarization. The inactivation can be reversed only by repolarizing the membrane to its negative resting potential, whereupon the channel switches to the resting state. This switch takes some time because channels leave the inactivated state relatively slowly (Figure 7-8).

Figure 7-8 Sodium channels remain inactivated for a few milliseconds after a depolarizing current. If the interval (Δt) between two depolarizing pulses (P₁ and P₂) is brief, the second pulse produces a smaller increase in g_Na because many of the Na⁺ channels remain inactivated after the first pulse. The longer the interval between pulses, the greater the increase in g_Na during the second pulse, because a greater fraction of channels will have recovered from inactivation during the period of repolarization and returned to the resting state when the second pulse begins. The time course of recovery from inactivation following repolarization contributes to determining the time course of the refractory period of an action potential.