7 Voltage Clamp

In the previous chapter, we covered ion flow and membrane potential changes that occur during the action potential in the neuron. We have this level of understanding about how ions move during the action potential because of a special technique called a voltage clamp experiment that was used in the 1950s. The voltage clamp method allows researchers to study voltage-gated ion channels by controlling the membrane potential of a neuron.

The Voltage Clamp Experiment

Initial Set-Up

To conduct a voltage clamp experiment, a portion of the axon, which would include the cell membrane and all the voltage-gated ion channels located there, is removed from a neuron and placed into a solution that mimics that of physiological extracellular solution. The ion concentrations across the membrane, as well as the electrochemical gradients, would remain the same.

Illustrated neuron showing segment of axon removed and put in a bath. Details in caption.
Figure 7.1. To conduct a voltage clamp experiment, a portion of the axon is removed from the neuron. The axon is placed in a special solution that is similar to physiological extracellular solution. ‘In Vitro Axon’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Measuring the Membrane Potential

The initial step in the voltage clamp method is to measure the membrane potential of the axon. A recording electrode is placed into the axon, and a reference electrode is placed into the extracellular solution. The voltage difference between these two electrodes is the membrane potential of the axon.

Illustrated axon with electrodes measuring membrane potential. Details in caption.
Figure 7.2. Measuring the membrane potential of the axon segment is the first step in the voltage-clamp experiment. The membrane potential is the difference in voltage between the intracellular recording electrode and the extracellular reference electrode. ‘Measure Membrane Potential’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Clamping the Voltage

The researchers running the experiment can set a desired membrane potential for the cell. The equipment then compares the desired membrane potential with the measured membrane potential from the electrodes. If these values differ, current is injected into the cell to change the measured membrane potential and make it equal to the desired potential.

Steps that occur during the voltage clamp experiment. Details in caption.
Figure 7.3. A desired membrane potential is set for the experiment. The voltage-clamp experimental equipment then compares the measured membrane potential with the desired potential. Current is then injected into the axon through a current-passing electrode to make the measured membrane potential equal to the desired potential. ‘Clamping Voltage’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Repeat

The equipment continues this cycle for the length of the experiment. It constantly measures and compares the actual membrane potential with the desired potential, and then uses current to correct any changes, “clamping” the potential at one value.

Steps that occur during the voltage clamp experiment. Details in caption.
Figure 7.4. The voltage clamp cycle repeats continuously. The actual membrane potential of the axon is measured, compared to the set desired potential value, and then current is passed into the axon to keep the actual membrane potential equal to the desired potential. ‘Voltage Clamp Cycle’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Voltage Clamp Experiment Example

At Rest

Let’s work through the system with an example. Here is an axon bathed in the extracellular solution. The resting membrane potential is measured at -65 mV.

Illustrated axon with resting membrane potential of -65 mV. Details in caption.
Figure 7.5. Measure the membrane potential. The membrane potential of this axon at rest is -65 mV. ‘Voltage Clamp Example at Rest’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Set Clamped Membrane Potential Value

For this experiment, the desired membrane potential value is 0 mV.

Illustrated voltage clamp experiment with a set clamped value of 0 mV. Details in caption.
Figure 7.6. Set desired membrane potential. The set value for this experiment is 0 mV. ‘Voltage Clamp Example Set Value’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Compare Actual and Set Membrane Potential Values

The equipment will determine that the actual membrane potential of the cell is not correct (-65 mV compared to 0 mV), so the cell must depolarize to reach the set value.

Illustrated voltage clamp experiment comparing actual and set membrane potential values. Details in caption.
Figure 7.7. Compare measured membrane potential to desired potential. The actual membrane potential of the axon is at -65 mV, so the cell needs to be depolarized to reach the desired potential of 0 mV. ‘Voltage Clamp Example Comparison’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Adjust Membrane Potential

To make the axon move from its resting membrane potential to 0 mV, the current electrode will pass positive current into the cell, depolarizing the cell until the membrane potential reaches the set value.

Illustrated voltage clamp experiment showing depolarization after inject. Details in caption.
Figure 7.8. Correct actual membrane potential. To depolarize this axon from rest at -65 mV to the desired clamp value of 0 mV, positive current will be injected into the cell. The membrane potential will then depolarize to 0 mV and remain there. ‘Voltage Clamp Example Current’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Ion Channels Continue to Function During Voltage Clamp

The important aspect of the depolarization seen in the example is that it is above threshold. Moving the membrane potential above threshold will activate the voltage-gated ion channels. Sodium channels will open immediately, and sodium will begin rushing into the cell. This influx of positive ions  would normally cause change the membrane potential to depolarize, but the voltage clamp equipment will measure the ion flow and inject a current of equal strength and opposite charge into the axon to maintain the membrane potential at 0 mV. This happens almost instantly and is a constant process, so as the ion flow changes, so does the injected current.

Animation 7.1. Clamping the cell at 0 mV will result in current being passed into the axon to depolarize the membrane potential. This depolarization is above threshold, so the voltage-gated ion channels in the membrane will be activated. Sodium will enter the axon through the open sodium channels. The voltage clamp equipment will inject current equal in strength and opposite in charge to the sodium influx in order to keep the membrane potential of the axon at 0 mV. The membrane potential will remain at 0 mV because the injected current offsets any change that would normally occur due to ion flow. ‘Voltage Clamp Sodium Flow’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License. View static image of animation.

Since the ion channels function as expected during the voltage clamp experiment, the voltage-gated sodium channels will inactivate, and the delayed voltage-gated potassium channels will open because, like the sodium channels, they are also activated when the membrane potential reaches threshold. This causes the ion flow to change from inward to outward. Normally, potassium efllux would cause a repolarization of the membrane potential, but the voltage clamp equipment will again inject a current that is equal in strength and opposite in charge to the potassium flow to keep the membrane potential steady at 0 mV.

Animation 7.2. The voltage-gated sodium channels will inactivate, and the potassium channels will open. Potassium will then flow out of the axon. Similar to the sodium influx, the voltage clamp equipment will inject current equal in strength and opposite in charge to the potassium efflux in order to keep the membrane potential of the axon at 0 mV. ‘Voltage Clamp Potassium Flow’ by Casey Henley is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License. View static image of animation.

Data Collection

Researchers can determine how much current is moving through the voltage-gated ion channels by observing how much current the equipment must inject into the cell to keep the membrane potential steady. If the equipment has to inject negative current in for 2 milliseconds, then the researchers know that positive ions were flowing in for 2 milliseconds. So the voltage-clamp set up allowed researchers in the 1950s to learn about how the voltage-gated ion channels were functioning during an action potential.

Key Takeaways

  • The membrane potential does not change during a voltage clamp experiment
  • Voltage-gated ion channels are still able to function normally and allow ion flow
  • If the clamped membrane potential is above threshold, the voltage-gated channels will act as if the cell is firing an action potential
  • The equipment must compensate for the neuron’s ion flow by injecting current into the axon. The amount of current needed to keep the membrane potential steady is equal and opposite to the current actually flowing in the cell

Test Yourself!

Additional Review

You conduct a voltage clamp experiment where you move the membrane potential from rest at -65 mV to 0 mV. You collect the following data:

Voltage clamp experiment results. Details in caption.
The membrane potential of a neuron is clamped at 0 mV. An initial inward current is seen, followed by an outward current.

Describe what causes the observed ion flow, including information about which ion(s) is/are moving and in which direction.

Answers

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