Biological Membranes - A. N. Ogurtsov 2012

Electrogenesis of Biomembranes
Action Potential
Voltage Clamp

The voltage clamp technique is a method in which the experimenter, rather than The Cell, sets the value of the Membrane Potential. Maintaining a constant voltage φ_M while studying currents across an excited membrane made it possible to:

1) eliminate capacitive currents

2) exclude Changes in the ionic conductances g_Na+ and g_K+ caused by variations in φ_М, and examine their changes during various phases of excitation development: g_i = f(t).

A constant potential difference between the inner and outer surfaces of the membrane is maintained using a specialized electronic circuit (Figure 117), the key element of which is an operational amplifier.

Class="center">

Figure 117 - Schematic diagram for studying membrane currents using the voltage clamp method: 1 - microelectrode; 2 - reference electrode; 3 - silver wire; 4 - DC voltage generator; 5 - ammeter

An operational amplifier (op-amp) is a DC amplifier with deep negative voltage feedback. The inputs of the operational amplifier receive the membrane potential φ_M = φ_int - φ_ext, which is the potential difference between the microelectrode inserted into the squid axon (1) and the reference electrode (2).

The output of the operational amplifier generates a voltage that compensates for changes in the transmembrane potential. This voltage is applied to the silver wire (3) running along the axon to ensure a uniform membrane potential difference across the entire fiber. The electronic circuit maintains the same potential at the output (inside the axon) as at the input of the operational amplifier, thereby keeping the membrane potential constant: φ_M = const.

Using the DC voltage generator (4), the input voltage to the operational amplifier can be stepped up, for example, to a level above the threshold. The electronic circuit will maintain this preset voltage throughout the experiment. The ammeter (5) measures the resulting membrane current flowing between the reference electrode (2) and the output electrode of the operational amplifier (3). In voltage clamp experiments, by Setting different constant values for the membrane potential φ_M, one can investigate how membrane current changes over time as excitation develops.

By convention, a current directed outward from the cell into the surrounding solution is considered positive, while a current from the surrounding solution into the cell is considered negative.

Experiments have shown that if the membrane potential φ_M is raised above the threshold, an inward current flows first (Phase 1), followed by an outward current from the cell (Phase 2) (Figure 118).

Figure 118 - Results of membrane current studies using the voltage clamp method

Experiments conducted by Hodgkin, Huxley, Baker, and Shaw demonstrated that Phase 1 of the membrane current is associated with an influx of sodium ions from the external medium (where the sodium concentration is higher) into the cell (where it is lower), whereas Phase 2 is explained by the efflux of potassium ions out of the cell.

In their experiments, Hodgkin and Huxley altered the ionic COMPOSITION OF THE external solution. They found that removing sodium from the extracellular medium abolished The first phase of the membrane current (the inward current). Consequently, the initial phase of Action Potential development is associated with an increase in membrane permeability to sodium ions. The influx of positive particles into the cell leads to membrane depolarization, causing its inner surface to become positive relative to the outer surface.

During the second phase, membrane permeability to potassium increases sharply, and positively charged potassium ions flow out of the cell while the sodium current diminishes.

The ionic mechanism underlying the action potential was definitively proven in an experiment by Hodgkin, Baker, and Shaw, in which the axoplasm of a prepared axon was replaced with an external solution, and the ionic composition of the extracellular fluid was adjusted to match that of normal axoplasm.

Following this Ion Exchange, the potential difference across the membrane reversed its sign. At rest, the inner surface of the membrane was now positively charged relative to the outer surface, while the action potential became negative.

Hodgkin and Huxley hypothesized that the selective changes in the ionic permeability of an excited membrane—first to Na⁺ and subsequently to K⁺—are due to the presence of specialized Ion Channels in the membrane (presumably pores formed by protein molecules).

They also postulated the existence of separate sodium and potassium channels that open and close as a Nerve Impulse passes through a given region of the membrane.

In the first phase, sodium channels open; In the second phase, potassium channels open. Conversely, sodium channels close first, followed by potassium channels. The opening and closing of ion channels are triggered by changes in the membrane potential.

One piece of evidence supporting the existence of ion channels in the membrane was the discovery of inhibitory substances that block ionic currents across the membrane.

For instance, tetrodotoxin, found in pufferfish, blocks The entry of sodium into the cell and thus disrupts nerve impulse transmission, which can be fatal. It has been proven that tetrodotoxin does not affect cell permeability to potassium, meaning that sodium and potassium ions actually pass through different channels. Due to its specific Structure, tetrodotoxin molecules selectively plug only the sodium channels.

By counting the number of tetrodotoxin molecules trapped in the membrane, researchers were able to determine the density of sodium channels. This varied among vertebrate nerve fibers, ranging from 3 to 75 channels per square micrometer of membrane area (by comparison, the number of phospholipid molecules is on the order of 2×10⁶ µm^-2).

A specific inhibitor of potassium channels, tetraethylammonium, was also discovered. If the membrane is treated with tetrodotoxin to block sodium channels, voltage clamp experiments reveal the disappearance of the first phase (Figure 118), whereas tetraethylammonium, which halts potassium Transport Across the membrane, causes the second phase to vanish.

Experimentally, the ionic current can be separated into individual components by substituting impermeable Choline cations for Na⁺ in the medium. In this case, the kinetic curve comprises only the K⁺ component.

Figure 119 shows the observed I(t) curves. When the axon is immersed in seawater, the total current I = I_Na + I_K is represented by curve 1. Upon replacing Na⁺ with choline, a pure potassium current is observed—curve 2. The difference between these two curves—curve 3—yields the sodium current I_Na.

Figure 119 - Membrane current and its components

Upon a rapid potential shift inside the fiber by +56 mV (membrane "short-circuiting"), the sodium conductance g_Na first increases rapidly from zero to 25∙10° Ohm^-1∙cm^-2 and then decreases (Figure 120).

Figure 120 - Changes in sodium and potassium conductance during membrane depolarization by 56 mV: solid lines show conductance during prolonged depolarization, dashed lines - during membrane repolarization after 0.6 and 6.3 ms

The potassium conductance increases slowly and reaches a steady level within 5 ms. Upon membrane repolarization, the sodium conductance decreases significantly faster than the potassium one.

Thus, it was established that the Generation of the action potential is driven by ionic fluxes across the membrane: first, an influx of sodium ions into the cell, followed by an efflux of potassium ions from the cell into the external solution, which is associated with Changes in membrane permeability for potassium and sodium ions.