Introduction

Hyperpolarization is said to be the final stage of an action potential after depolarization and repolarization in action potential, respectively. Many diseases and conditions may arise from dysfunctions and mutations in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. These disorders include Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis among neurodegenerative disorders. Epilepsy is also seen due to these HCN channel mutations.

Hyperpolarization Vs Depolarization

Hyperpolarization can be thought of as the opposite of depolarization. Hyperpolarization occurs as an overshoot during repolarization. 

In depolarization, the cell’s membrane potential becomes less negative up to a membrane potential of +40mV.  

In hyperpolarization, on the other hand, the cell’s membrane potential becomes more negative, this makes it more difficult to elicit an action potential as we are deviating away from the action potential threshold. 

This phase of hyperpolarization occurs after repolarization which causes the membrane potential to drop back down to -70mV from +40mV. Whereas depolarization is caused by sodium ions influx through voltage-gated sodium ion channels, hyper-polarization is caused by potassium ions efflux or calcium ions influx through their respective voltage-gated channels. This potassium ion efflux occurs down its concentration and electrostatic pressure gradient. 

Similarly, calcium ion and sodium ion influx through sodium ion and calcium ion channels inhibit hyper-polarization. Similar to repolarization, hyperpolarization is achieved through potassium ion efflux through voltage-gated potassium ion channels. However, another method by which hyperpolarization is achieved is through chloride ion influx through chloride ion channels.

Voltage gated Ion Channels

The main voltage-gated channels involved in actions potentials are sodium ion, potassium ion and chloride ion channels. These ions are attracted to or repulsed by these ion channels. The channel releases its water molecule, and the ion is able to pass through the pore. A stimulus is required to open and close these voltage-gated channels. 

Channel gating is when the channel is able to reopen right away after closing. Channel inactivation is the opposite when the channel is closed and cannot reopen after.  

Sodium Ion Channels

On the opening of the voltage-gated sodium ion channels, the depolarization of the neurons creates a current feedback loop. This is called Hodgkin’s cycle. The neuron only elicits an action potential if the depolarization reaches the threshold potential. The permeability of the neuron to sodium ions is 10 times greater than that to potassium when all the sodium ion voltage-gated channels are open. On reaching a potential of +40mV, the sodium ion channels close and voltage-gated potassium ion channels open. This next phase is called repolarization. 

Repolarization

Repolarization causes the membrane potential to drop back down to a negative value. The repolarization continues all the way until the cell membrane potential reaches -75mV. This voltage of -75 mV is the equilibrium potential of potassium ions. At -75mV the neuron is hyperpolarized. Following hyperpolarization, the potassium ion channels close and the cell returns to its resting membrane potential of -70mV.

Patch Clamping Method

A method by which hyperpolarization can be measured is patch clamping. This is done using a patch pipette. A patch pipette is a glass micropipette that has a diameter of 1 micrometre. The point of entry of the current is through a small patch containing a few ion channels. The membrane potential is maintained by a voltage clamp and amplifier, this voltage clamp is then able to measure small current flow changes. Hyperpolarization is measured either as an increased outward current or decreased inward current.  

Depolarization

Prior to hyperpolarization and in the first stage of an action potential, we have depolarization. Initially, the cell is at its resting membrane potential in which it has a charge of -70mV.  In depolarization, the cell’s negative charge becomes less and the positive charge increases.  This change is temporary only during the phase of an action potential. The cell polarity reverses by the end of the depolarization phase of the action potential with the cell becoming positively charged. The reason for this reversal in polarity and change in charge is due to the influx of sodium ions through voltage-gated sodium channels. 

Refractory period

When a neuron is in a hyperpolarized state it is also in a 2-millisecond refractory period. During the 2 second refractory period after hyper-polarization, the membrane potential overshoots past the resting potential and then returns to its resting membrane potential once again thereafter.

Read more about Repolarization

The refractory period otherwise called refractoriness of a neuron is the behaviour of an auto wave to not respond to stimuli. It can also be called the recovery time.  It can also be defined as the time it takes for a membrane to get ready for its next stimulus after reaching its resting membrane potential. 

There are two types of refractory periods. 

The absolute refractory period is associated with depolarization and repolarization. The relative refractory period on the other hand is associated with hyperpolarization. The absolute refractory period is caused by the inactivation of the sodium channels which initially caused the depolarization, the inactivation of these channels is in effect up until the membrane hyperpolarizes. 

On hyperpolarization these sodium ion channels are deactivated on closing and are once again are able to open on receiving a stimulus. This absolute refractory period corresponds to the entire action potential phase. Then following repolarization and the absolute refractory period we have the relative refractory period. As the membrane potential is more negative than the resting membrane potential, it becomes more difficult to reach the threshold potential. The resting potential is achieved by sodium-potassium ATPases to its resting potential of -70mV. Once the resting membrane potential is again achieved, the relative refractory period is over.

Epilepsy 

Epilepsy is one of the most common and severe neurological disorders. Epilepsy is characterized by recurrent spontaneous seizures. Approximately 1/3 of those with epilepsy do not react appropriately to treatment. The syndromes can classify as focal (partial) and generalized syndromes. 

HCN channels in Epilepsy

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are one of the most important components to study regarding epilepsy. These channels modulate rhythmic activity, synaptic transmission, cellular excitability, and dendritic integration to name a few. 

There are 4 types of HCN channels namely HCN1, HCN2, HC3 and HC4. Each HCN channel has a specific voltage characteristic and cAMP sensitivity. These HCN channels are important in both the neurons in the brain and myocytes in the heart. Reverse voltage dependant channels such as hyperpolarization-activated cyclic nucleotide channels are dependent on membrane hyperpolarization. Cyclic adenosine monophosphate (cAMP) is one of the few transduction cascades that are able to regulate it. In a few cardiac diseases, HCN channels are important in the generation and conduction of the electrical impulses. These HCN-gated channels are said to be the pacemakers of the brain. Hence dysfunctions are commonly are the underlying cause of epilepsy. 

Genetics 

Epilepsy is hypersynchronous network activity in the brain. Recent studies indicated sequence variations in HCN1 and HCN2 genes to have an association with epilepsy. One of these variations is a triple proline deletion in HCN2. This triple proline is important in channel function. Patients with febrile seizure syndromes are more commonly found to have this triple deletion. In the heart, these HCN channels conduct If currents, in the brain they conduct Ih currents. Studies reveal mutations in the HCN1 and HCN2 to have an association with epilepsy, in specific these channel mutations are normally involved with Genetic Generalized Epilepsy (GGE). An A881T mutation in HCN1 and R5271 mutation HCN2 in a GGE family was found. One study found that 2.4% of febrile seizure syndrome patients had the triple proline deletion in the HCN2 channel, however, this value was 0.2% in unaffected control cases. This however is expected in susceptibility genes. 

Mouse Experiment

One experiment that indicated significant evidence of altered HCN channel function to epilepsy is that of the HCN2 knockout mouse experiment. This mouse is found to have bilateral synchronous SWD, this is a hallmark of absence epilepsy which is a very common seizure in GGE. The apathetic mouse is similarly found that to exhibit this SWD phenotype. A spontaneous Hcn2 truncation is seen here. The knockout mouse HCN2 thalamocortical neurons are more greatly hyperpolarized and have a more prominent burst firing phenotype in relation to controls consistent with Ih reduction. The availability of Calcium ion T-type channels is increased by the HCN2 knocking out TC neurons more hyperpolarized resting membrane potential.

In another experiment looking at rat models, we get some evidence of the role of these HCN channels in the absence of seizures. These are the Generalized Absence Epilepsy Rat from Strasburh and Wistar Albino Gilaxo rat models. A reduction in Ih was seen in the L2-3 pyramidal neurons and slower activating HCN channels was seen in these models. Ih s are also important in the membrane leak at resting membrane potential, this is especially the case in dendritic arbour. In dendritic arbour HCN1 channel expression is the greatest. L5 pyramidal neurons in these 2 models also show this reduction in the expression of these HCN1 channels. This loss of HCN1 function increases back-propagating action potentials which may increase excitability in these distal dendrites.

HCN Channels in other Neurodegenerative Disorders

HCN channels are said to play a role in the development of other neurodegenerative diseases including the likes of Alzheimer’s disease, spinal muscular atrophy (SMA), Parkinson’s disease and even amyotrophic lateral sclerosis. We must however not take any credit away from the distinct pathological mechanism underlying these diseases rather we must bear in mind that there is increasing evidence of HCN channel and other ion channels contributing to these disease mechanisms. When the discussion of Parkinson’s disease arises, HCN function and regulation is essential to observe. 1-methyl-4-phenylpyridinium (MPP⁺)-induced toxicity may be due to I_h dysfunction. This may one of the pathogenic mechanisms found in Parkinson’s disease. 

Other contributions of the HCN channel in Parkinson’s disease include that its function in neuroinflammation and depression hence resulting in non-motor symptoms and neuroinflammatory problems. These HCN channels are also found to contribute to Alzheimer’s disease pathogenesis and symptoms. One example of this is its role in regulating Beta-amyloid peptide formation. These channels in addition play a vital role in learning memory and learning.

Conclusion

Action potentials are the most fundamental aspects of neuronal function. Action potentials consist of a number of phases. 

Action potentials start off with depolarization mediated by the opening of sodium ion channel through which an influx of sodium cations raises the membrane potential from its resting membrane potential of -70mV to +40mV. 

On reaching a potential of +40mV, these sodium ion channels close and voltage-gated potassium ion channels open causing an efflux of potassium ions from the neuron. This phase is called repolarization which drops the membrane potential to drop back down to its resting membrane potential of -70mV. 

The membrane potential then briefly overshoots past the resting membrane potential of -70mV to a membrane potential of about -75mV. On reaching this membrane potential of -75mV, the neuron is said to be in a hyperpolarized state. The neuron then returns to its resting membrane potential by the closing of potassium ion channels and is ready to elicit its next action potential. This Hyperpolarization although a rather short phase in an action potential is proven to play a very vital role and the slightest disturbance in the process is seen to have catastrophic effects. 

The HCN channels are the key structures and disturbances of this channel are seen to have various pathological consequences. 

Dysfunctions in these channels contribute to various neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and more. 

Apart from these neurodegenerative diseases, HCN channel dysfunctions and mutations in addition are found to contribute to epilepsy.

Frequently Asked Questions

What is hyperpolarization?

The term hyperpolarization is used to describe a state when the membrane potential becomes more negative than the resting membrane potential. It happens towards the end of an action potential.

What happens during hyperpolarization?

During hyperpolarization, potassium ions keep leaking out of the cell via potassium channels. This diffusion of potassium ions (positively charged ions) into the extracellular space makes the membrane potential more negative.

Which type of channels are involved in hyperpolarization?

Hyperpolarization happens due to the diffusion of potassium ions via potassium channels. These are voltage-gated channels that respond to the changing membrane potential. 

How hyperpolarization is reversed?

Hyperpolarization is reversed to achieve the resting membrane potential with the help of ATP dependent sodium-potassium pump, also known as sodium-potassium ATPase. It pumps sodium ions out of the cell while bringing potassium ions into the cell, both against the concentration gradient. This helps in achieving the resting membrane potential.

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