Effects of neurotransmitters on axonal KV7 channel function in hippocampal neurons

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.

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.

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.