Hodgkin-Huxley Action Potential Notes

The Action Potential

• The Action Potential (AP) is the unit of neural communication.
• It requires differences in the ionic composition of intracellular and extracellular fluids (concept from Chapter 3).
• These ionic differences are a source of energy for electrical signaling.
• The AP is characterized by a reversal of membrane potential—from very negative to very positive.
• It enables information to be conveyed over long distances along neurons.

The Giant Squid Axon and the HH Pioneers

• Masterminds: Alan Hodgkin and Andrew Huxley.
• In 1939, they observed the action potential in the giant squid axon using intracellular recording.
• The squid axon provided a suitable preparation (large diameter) for measuring resting and action potentials.
• Experimental setup involved an Axon, an Amplifier, Reference electrode, Recording electrode, and Stimulus.
• Key finding: the axon’s AP could be measured intracellularly and is a reversible change in membrane potential.
• They demonstrated that the AP could be studied with intracellular techniques that reveal how voltage changes relate to ionic movements.

Intracellular Recording and Threshold

• Method: measure the voltage difference between the inside and outside of a neuron (intracellular recording).
• To elicit an AP, inject current to drive the neuron to threshold, initiating the AP.
• This intracellular recording approach was fundamental for understanding the shape and mechanics of the AP.

The Ava Case (Hyperexcitability and Threshold)

• Researchers recorded intracellularly from Ava’s neurons and a control neuron.
• They applied two stimuli: one subthreshold and one that normally reaches threshold.
• Ava’s neurons were hyper-excitable and firing constantly (pattern typical of stroke).
• Resting Membrane Potential (RMP) was very high, so little added current was needed to reach threshold.
• If constant current did not reach a true threshold, seizures could occur due to persistent firing.
• Conceptual takeaway: elevated RMP lowers the threshold for triggering an action potential.

The Challenge: What Causes the Inside to Become More Positive?

• When sufficient charge is delivered, the inside of the cell becomes more positive.
• It was unclear which ions were responsible for the changes during the AP.
• Possibilities discussed included:
  • Cations moving in or anions moving out to produce depolarization.
  • After the peak, why does the potential become more negative again? Potential explanations included different ions moving out or in, or ions entering/leaving in a sequence.
• Limitation: With intracellular measurements alone, isolating the activity of individual ions at each step of the AP was not possible because changing ion concentrations also changes membrane potential.

The Voltage Clamp: Pausing the AP to Measure Currents

• The voltage clamp technique pauses the AP at a chosen voltage by injecting countercurrent to keep the membrane at a fixed voltage.
• It allows changing intracellular and extracellular ion concentrations while holding the voltage steady.
• What is measured: the current you inject to hold the voltage (the countercurrent) is essentially the net ionic current across the membrane for that voltage.
• This method provides a controlled way to study how different voltages drive ion movements.

The Neuron Voltage Clamp (Historical Context)

• Invented in the 1940s and adapted by Hodgkin and Huxley around 1950.
• Basic setup: outside is the ground/reference electrode; intracellular electrode records voltage; a voltmeter compares actual Vm with the commanded voltage (V_cmd).
• If Vm ≠ Vcmd, a current is injected to correct and bring Vm to Vcmd.
• The injected current is the ionic current across the membrane for that voltage.
• This arrangement allows precise measurement of how much current is needed to maintain a given membrane potential.

Dissecting Ion Contributions: Sodium vs Potassium

• Goal: isolate the contributions of different ions to the currents underlying the AP.

• First manipulation: remove Na+ from the extracellular solution.

  • Questions asked: How much current is needed to hold the membrane at each voltage? Why does this change with voltage? How quickly does the current flow? Is this dependent on voltage? What does this tell us about the inside of the cell? Is the current inward or outward?
  • Observation: without extracellular Na+, the inward Na+ current is eliminated; remaining outward currents reflect other ions (primarily K+).

• Relevance: By removing specific ions, researchers could infer which currents are carried by which ions and how these currents vary with voltage.

• Second manipulation: remove K+ from the extracellular solution.

  • Observations: with reduced/absent K+, the current associated with K+ outward flow diminishes; Na+ current becomes more evident (inward) and Na+ channels inactivate quickly.
  • Na+ current is inward (driving the depolarization) and rapidly inactivates; K+ contributes a slower, outward current that repolarizes the membrane.

I–V Plots: Revealing Two Distinct Currents

• Systematically vary the test potential and measure the peak inward and outward currents at each voltage.
• Construct an I–V plot to show the current-voltage relationship.
• Key finding: there are two components of current during the AP:
  • An early inward current (inward Na+ current).
  • A late outward current (outward K+ current).
• These two components have distinct voltage dependencies and kinetics.

What This Means: Two Voltage-Gated Mechanisms

• Conclusion: There must be two different voltage-gated mechanisms controlling the AP.
• Na+ component: fast inward current that activates quickly and tends to depolarize the membrane; Na+ channels inactivate rapidly.
• K+ component: slow outward current that does not inactivate in the same way, supporting repolarization.
• This led to the understanding that two gating processes counteract each other during the AP: rapid Na+ activation/inactivation and slower K+ activation.

The Na+ Channel Discovery and Patch Clamp

• It took about 26 more years to prove the Na+ voltage-gated channel existed and carried the fast inward current.
• The Patch Clamp technique was developed as a more specific form of voltage clamp, enabling recording from individual ion channels one at a time.
• This allowed direct observation of single-channel behavior and detailed characterization of channel kinetics.

The Action Potential Timeline and Membrane Phases

• The classic recording shows resting membrane potential around a negative value, followed by an action potential rising to a positive peak, then repolarization and hyperpolarization.
• During the waveform, the membrane passes through:
  • Resting membrane potential (Vm resting)
  • Rising phase (depolarization)
  • Peak (often near or above 0 mV in many neurons)
  • Falling phase (repolarization)
  • Absolute refractory period (no second AP can be elicited regardless of stimulus)
  • Relative refractory period (a stronger stimulus can elicit a second AP, but it is harder than at rest)
• The figure also emphasizes the general idea of driving forces during the AP.
• Question highlighted: Which ion has the greatest driving force at each point in the AP? Driving force depends on the difference between the membrane potential and the ion’s equilibrium potential: $DF<em>i = V</em>m - E_i$.
• Early in the AP, the driving force for Na+ is large and pushes depolarization; later, as the Na+ current inactivates, K+ conductance increases and its driving force dominates to repolarize.

Resting Membrane Potential: Leak Dominance

• Resting membrane potential is largely determined by leak currents.
• Most of the leak current is carried by K+ ions; there is a smaller Na+ leak.
• At rest, voltage-gated ion channels are closed, so the membrane is mostly permeable to K+ through leak channels.

Key Formulas and Values Mentioned in the Transcript

• Change in membrane potential during AP reversal:
  • The AP peak can exceed the resting potential by about $ext{40 mV}$, i.e., $\big| ext{AP peak} - V_{rest}\big| \approx\, 40~ ext{mV}$.
• Resting membrane potential: $V_{rest} \, \approx -65 ext{ to } -70 \, \text{mV}$.
• Driving force for ion i: $DF<em>i = V</em>m - E<em>i$, where $E</em>i$ is the equilibrium (Nernst) potential for ion i.
• Currents used in the HH framework: denoted as $I<em>{Na}$ (sodium current) and $I</em>{K}$ (potassium current), with their distinct activation/inactivation properties and kinetics.
• Command voltage in voltage clamp is denoted as $V<em>{cmd}$; the actual membrane potential is $V</em>m$; the injected current to hold the potential is the measured ionic current.
• Two main gating events: fast Na+ activation with rapid inactivation; slower K+ activation that does not inactivate on the same timescale.

Practical and Conceptual Takeaways

• The AP arises from tightly regulated, voltage-dependent ion conductances, not a single ion movement.
• Intracellular recording showed that ions are involved, but only later did voltage clamp reveal the separate ion currents and their dynamics.
• The voltage clamp was a pivotal methodological advance, enabling the isolation and quantification of ionic currents across the membrane at fixed voltages.
• The discovery of Na+ voltage-gated channels and the Patch Clamp technique were fundamental milestones for neurophysiology and the study of ion channels.
• Understanding the resting state, AP, and refractory periods provides a framework for how neurons code and transmit information.