Effects of neurotransmitters on axonal KV7 channel function in hippocampal neurons

Lead Research Organisation: University College London


The brain is primarily constituted of nerve cells (neurons). Neurons contain a cell body (soma). There are many protrusions from the cell body. The vast majority of these are known as dendrites. Dendrites receive analogue signals (post-synaptic potentials) from other neurons and relay these to the cell body. The cell body processes and integrates these signals and passes them onto the axon, a specialized structure that emerges from the cell body. The primary role of the axon is to convert these analogue signals into digital, all or none signals, known as action potentials. Action potentials travel along axons and cause neurotransmitter release from these. The neurotransmitters in turn bind to proteins, receptors, located in dendrites and generate post-synaptic potentials. These are the fundamental cellular mechanisms that underlie many normal brain functions such as learning and memory (cognition). It is important to understand how these cellular mechanisms are regulated as dysfunction of these can lead to neuronal disorders such as mild cognitive impairment (MCI) and dementia.

In addition to receptors, neurons contain specialized proteins, ion channels, which form pores in the membrane. These are permeable to particular ions such as potassium, sodium and calcium. Some open and close in response to small changes in voltage across the cell membrane and are known as voltage-gated ion channels. Others are activated by ligands (neurotransmitters) binding to them - ligand-gated ion channels.

We are interested in a particular type of voltage-gated ion channel, the Kv7 channel, which is permeable to potassium ions only and is located principally in axons. We have recently shown for the first time that in the hippocampus a key area of the brain involved in learning and memory, axonal Kv7 channels are open continuously at rest. Since potassium ions flow out of cells, these channels form a constant inhibitory current. This makes it more difficult for action potentials (which are large positive voltages or spikes) to be generated. This effect of Kv7 channels is likely to help a neuron maintain its normal activity. Indeed this might explain why mutations in Kv7 channels in humans lead to epilepsy.

New evidence suggests that axons can contact each other and that neurotransmitter receptors may also be present on axons. Our new data show that axons that release the neurotransmitter, acetylcholine, are closely associated with hippocampal axons. Further, our data show that these hippocampal axons also express receptors for the major excitatory neurotransmitter in the brain, glutamate. It is unknown though if acetylcholine and glutamate, by binding to their respective receptors, can activate signaling proteins within a neuron and lead to changes in the properties of axonal Kv7 channels. More importantly, we do not know how this might alter information processing within axons. In this study, we aim to investigate these problems by using state-of-the art methods such as making electrophysiological recordings of currents generated from Kv7 channels present in single hippocampal neurons and using specific reagents (some of which we have designed ourselves) to study the function of axonal Kv7 channels.

Since a decline in Kv7 channel function results in loss of hippocampal-dependent learning, loss of cholinergic neurotransmission is associated with cognition related disorders such as mild cognitive impairment, dementia and Alzheimer's disease, and since axon loss occurs during normal aging, it is important to understand the factors involved in axon information processing. This could ultimately lead to better therapies for disorders such as mild cognitive impairment and Alzheimer's disease, which are prominent in the elderly population and for which at present there are few treatments available.

Technical Summary

The voltage-gated K+, Kv7 channels underlie a non-inactivating current, the 'M-current', in peripheral and central neurons. This current is modulated by neurotransmitters, particularly acetylcholine acting on muscarinic G-protein coupled receptors (GPCRs). New evidence shows that Kv7 channels are mainly located in the axon initial segments (AIS) of neurons. We have recently shown that these channels are crucial for determining the action potential threshold and thus action potential initiation, neuronal firing patterns and excitatory post-synaptic potential (EPSP)-spike coupling in hippocampal CA1 pyramids. The AIS, however, is a specialized region of the cell that is densely packed with proteins and lipid rafts. It is unknown if these axonal Kv7 channels are regulated by GPCRs activated by neurotransmitters (e.g. acetylcholine and glutamate) and, if so, what second messenger systems may be involved. These are the main objectives of this proposal.

We will investigate these aims using hippocampal dentate gyrus granule cells as 1) our data show that, in contrast to hippocampal CA1 pyramids, these neurons have solely axonal Kv7 channels; 2) we find that cholinergic boutons are very closely associated with granule cell axons (mossy fibres); and 3) mossy fibres express metabotropic glutamate receptors. We will predominantly use electrophysiology, immunohistochemistry and pharmacology. The data generated will provide novel information on how axonal Kv7 channels are modulated by cholinergic and glutamatergic inputs, the second messenger systems involved and the impact of this modulation on neuronal excitability. Further, since Kv7 channels and the dentate gyrus play critical roles in hippocampal-dependent spatial learning, cholinergic dysfunction underlies cognitive disorders associated with aging and axons degenerate during aging, the findings will provide vital information on the processes underlying age-related disorders such as mild cognitive impairment.

Planned Impact

Understanding axon information processing is critical for comprehending how the brain works. Recent advances indicate that axons express a variety of proteins, including ion channels, which have a significant impact on axonal signal transduction. Further, there is now some evidence that neurotransmitters might be able to influence their properties, representing another means by which axonal conduction is regulated. In this project, we will investigate how the function of the potassium, Kv7 channels, which are predominantly located in axons in the central and peripheral nervous systems, are modulated by neurotransmitters. The results will yield crucial information on Kv7 channel plasticity, neurotransmitter receptor function and axonal signalling. Since axons shrink with normal aging leading to cognitive disorders (e.g. mild cognitive impairment (MCI)), as compromised axonal conductance is a feature of many neurodegenerative disorders (e.g. multiple sclerosis and Alzheimer's disease) and because Kv7 channel mutations have been associated with epilepsy and deafness, there are likely to be numerous beneficiaries beyond the immediate K+ channel field. These include the academic (fellow scientists and clinicians) and commercial sectors (the pharmaceutical industry), the wider public (including charities such as Age UK, Multiple Sclerosis UK and Epilepsy Research UK) and the UK economy.

The UK parliament (www.parliament.uk) has forecast the aging population (>65 years) to increase by 50% within the next twenty years and to double by 2050. Improving treatments for age-related and neurodegenerative disorders is essential to ensure that the National Health Service and other welfare systems are not strained. Our work will provide a better understanding of the impact of cognitive/neurodegenerative disorders on brain function and identify new potential therapeutic targets for these. This is particularly important as the current treatments for MCI and for neurodegenerative disorders such as dementia and multiple sclerosis are not very effective and are associated with many side effects. Better treatment of these disorders would ensure that the affected individuals would be able to lead normal, independent lives, thus reducing the stress on public services and enhancing the UK's economy.

Further, Kv7 channels and many neurotransmitter receptors (particularly the cholinergic receptors whose function we will be exploring) are recognized targets for the treatment of age-related neurological disorders such as dementia, Alzheimer's disease and neuropathic pain. Retigabine, a Kv7 current enhancer, has recently been approved for the treatment of partial seizures and is currently in clinical trials for neuropathic pain therapy. Since there is very little information available on the impact of neurotransmitter receptors on axonal ion channels such as Kv7 channels and the effects of this on axon signal transduction, this work will be of immense interest to the pharmaceutical industry and could help them more effectively evaluate the potential of neurotransmitter receptors and axonal ion (Kv7) channels as therapeutic targets, leading to perhaps better treatments.

We will be presenting our work at major national and international conferences (including those arranged by charities such as Epilepsy Research UK which are attended by many health professionals) at regular intervals within 1-2 years. It is, therefore, likely that the influence our work has on academics and commercial (pharmaceutical industry) can be quite rapid (within 2-3 years). The influence that this may have on the UK economy is likely to be more long-term (within the next 10 years or so).

Finally, this project will also fund a young, talented electrophysiologist and help them advance their research related skills in electrophysiology and imaging as well as more generic skills such as computing, presentation and writing report skills that are required in all sectors.


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Description Neurons are brain cells. They have a cell body (somata) and two main types of processes (axons and dendrites). Dendrites receive information from other neurons and process this information. This is then relayed to axons via the cell body. Axons will then generate signals (action potentials) that will cause the release of chemicals (neurotransmitters) from nerve endings (synaptic terminals). Understanding how axons process information that leads to release of neurotransmitters is important as axon damage and re-routing is a hallmark of many disorders such as dementia, Alzheimer's disease, multiple sclerosis, epilepsy and Huntington's disorder.
We have discovered that acetylcholine, a neurotransmitter that plays a fundamental role in cognition, can significantly alter axonal information processing in the hippocampus, a key brain region involved in learning and memory. This is the first time that this has been demonstrated. We have also made significant progress in understanding the cellular mechanisms by which acetylcholine does this. This may lead to novel therapeutic targets for the treatment of cognitive disorders. This is necessary as acetylcholinesterases, which prevent the breakdown of acetylcholine and are currently used for the treatment of mild Alzheimer's disorder, are not suitable for all patients.
Exploitation Route We showed that acetylcholine and the mGluR agonist, DHPG, cause a long-lasting reduction in the action potential threshold in adult dentate gyrus granule cells. The effects of acetylcholine occurred by acting on the muscarinic M1 receptors located on axons. Similarly DHPG also caused alterations in action potential threshold by acting on mGluR1/5 receptors on axons. We demonstrated that these receptors activate axonal T-type Ca2+ currents to raise intracellular axonal Ca2+ which then inhibits axonal KV7 channels. This mechanism is distinct from that which occurs in the somata, whereby muscarinic receptors activation leads to a reduction in PIP2 levels and inhibition of KV7 channels. This work was published in Neuron.

We have since also determined that muscarinic M1 receptors enhanced the levels of the kinase Ca2+/Calmodulin-dependent kinase II (CaMKII) to cause a shift in axonal T-type Ca2+ currents. CaV3.2 subunits underlie this T-type Ca2+ current and we have started to understand which residues on these CaV3.2 subunit are phosphorylated by CaMKII. Some of this work was presented at FENS (Sitnikov et al., FENS, 2018).

Finally, we have also shown that KV7 currents are present on the giant synaptic terminals of granule cell axons, the so-called 'mossy fiber terminals'. By making whole-cell and outside-out patch electrophysiological recordings from these boutons, we demonstrated that the KV7 current in these boutons has a very unusual negative half activation voltage (-70 mV) and is active at rest. The main function of this KV7 current is to restrict Ca2+ influx through voltage-gated Ca2+ channels into the terminal. Consequently, the Ca2+ influx during an action potential is reduced when these channels are active and this results in reduced action potential-dependent synaptic release. By making paired recordings from mossy fiber bouton terminals and CA3 pyramidal neurons (on which the terminals synapse), we showed that action potential dependent synaptic release was enhanced in the presence of KV7 current inhibitors. This work has now been accepted for publication by Communications Biology.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://iris.ucl.ac.uk/iris/browse/profile?upi=MSHAH22
Description Location-dependent effects of neurotransmitters in neurons
Amount £198,185 (GBP)
Funding ID RPG-2020-102 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2020 
End 10/2023
Description Project grant
Amount £361,121 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 07/2018
Description Royal Society International Exchange Award
Amount £6,000 (GBP)
Funding ID IE111246 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 03/2013
Description UCL - Peking University Strategic Partner Fund
Amount £10,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 09/2019 
End 10/2020
Title Ankyrin G binding peptide 
Description Ankyrin G binding peptide is a useful tool for selectively inhibiting axonal KV7 channels. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact We will publish the results generated using this soon. 
Title Model for mossy fiber bouton terminal 
Description Based on our experimental findings, we collaborated with Dr. M. Migliore to generate a computational model that can simulate how neuronal excitability alters when particular K+ or Ca2+ current densities or biophysical characteristics are altered. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2019 
Provided To Others? Yes  
Impact This work has been accepted for publication by Communications Biology, a new journal by the Nature group. 
URL https://senselab.med.yale.edu/modeldb
Title Mossy fibre bouton model 
Description This is a model of a mossy fibre synaptic terminal incorporating new ion channel biophysics data on KV7 channels. The mossy fibre bouton is a synaptic terminal that has been used extensively to study synaptic transmission. We made electrophysiological recordings to show that particular K+ channels, the KV7 channels, are present prominently in these terminals and are important for regulating their excitability and synaptic release. To better understand how they do this, we generated this computational model. 
Type Of Material Computer model/algorithm 
Year Produced 2019 
Provided To Others? Yes  
Impact This model will be useful for others who wish to test out their hypothesis on how ion channels affect synaptic release from terminals. Experiments to do this are very challenging and require considerable number of animals. Hence, this computational model will be useful for testing out hypothesis, thereby reducing pilot experiments that may need to be done and the animals required for these experiments. 
URL https://senselab.med.yale.edu/modeldb
Description Computational Modelling 
Organisation National Research Council
Department Institute of Genetics and Biophysics (IGB)
Country Italy 
Sector Public 
PI Contribution We have obtained electrophysiological data from the dentate gyrus axon terminals (mossy fiber boutons) that synapse onto CA3 pyramidal neurons. We found KV7 channels present here. They had very unusual biophysical properties. We then used pharmacological tools to study the effects of KV7 channels on the intrinsic excitability of the terminals. Some unusual effects were detected and we, therefore, formed collaboration with Dr. Migliore at the Institute of Biophysics to generate a computational model to try to explain some of our findings.
Collaborator Contribution They have generated a novel computational model for axon terminals. We have used this model to understand the effects that we observe.
Impact We are writing up the manuscript, which will be submitted in the next few weeks. This is a multi-disciplinary collaboration as they used mathematical equations to generate the computational model. We used electrophysiological recordings from boutons present in rat brain slices.
Start Year 2017
Description GPCR binding partners 
Organisation University College London
Department Neuroscience, Physiology & Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have advanced our understanding of the signalling mechanisms activated by G-protein Coupled Receptors in neuron axon initial segments. We have done this work using custom-made peptides, pharmacological agents, electrophysiology and Ca2+ signalling in neurons present in brain slices. The team led by Matt Gold is performing biochemical and molecular biology assays to help us better understand whether GPCRs bind to particular partners and how these may then interact with others to activate ion channels in axons.
Collaborator Contribution As stated above we have done functional assays using adult mouse brain tissue. Matt Gold's team are now doing molecular biology/biochemical assays.
Impact We have co-applied for a BBSRC project grant
Start Year 2022
Description Nuffield Foundation Studnt 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A Nuffield Foundation student who was an A-level student interested in Neuroscience spent 6 weeks in my lab. She was trained by my Ph.D. student in immunohistochemical methods. She then wrote a report and did a presentation of the work at her School and also at a conference organised by the Nuffield Foundation.
Year(s) Of Engagement Activity 2015
Description Outreach afternoon for Schools 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We presented two posters sharing information on what we do in the lab.

A-level students visited the lab and expressed an interest to study science.
Year(s) Of Engagement Activity 2014