Structural studies into human muscle nicotinic acetylcholine receptors

Muscle type nicotinic acetylcholine receptors (nAChR) are the key components of the neuromuscular junction that receive the acetylcholine signal from the motor neurone to initiate muscle membrane depolarisation and muscle contraction. They are the archetypal members of the pentameric ligand-gated ion channel family that also includes receptors to other neurotransmitters such as GABA, serotonin, and glycine. The early discovery of these receptors helped develop many of the methods used to study ion channels, which led to much of our understanding of how ion channels function.

Muscle type nAChR are also some of the most complex members of the pentameric ligand-gated ion channel family and is made up of 4 different subunits - 2 alpha1, 1 beta1, 1 delta and either a gamma subunit in foetal receptors, or an epsilon subunit in adult receptors. Expression of the adult receptor initiates a few weeks before birth, after which it is selectively incorporated into the neuromuscular junction. Abnormalities in this receptor lead to myasthenic syndromes where patients can suffer from muscle weakness of varying severity from foetal akinesia and death in utero to fatigable muscle weakness that causes a mild lifelong disability. So far, very little information is available on the structure of the human muscle nAChR receptors, with most of the studies carried out on the electric organ receptor of the electric ray. While this ortholog has similar subunit composition to human foetal nAChR, it nevertheless does not fully recapitulate the human protein's behaviour and cannot be reliably used to predict whether genetic variants identified in patients cause disease. This is because disease causing variants are most commonly found in the epsilon subunit, which is not present in the electric ray. These orthologue structures are also not accurate enough for drug discovery, where structure based approaches greatly accelerate the development of new medicines.

The aim of this proposal is to obtain a detailed picture of how human muscle type nAChRs work by resolving structures of the proteins at near atomic resolution, followed by detailed assessments of how the protein works. We have already established protein production and purification methods for both adult and foetal receptors, which we used to produce a promising preliminary cryo-electron microscopy dataset of the adult receptor at 9 Å resolution. To produce higher-resolution structural information, we will refine these methods and apply a variety of other strategies to trap it in different functional states, including using disease-causing variants of the receptor and using drugs that are known to target the receptor.

Building on these results, we will examine the function and responses to different stimuli with structure-guided mutations. These will decipher the channel's structure-function relationship and the molecular mechanisms that underlie how it works. This information will ultimately help predict whether genetic changes observed in patients are likely to cause disease, as well as assist in designing new drugs that target this protein to treat myasthenia and other forms of neuromuscular diseases.

The muscle type nicotinic acetylcholine receptor (nAChR) is the archetypal pentameric ligand-gated ion channel (pLGIC) that is essential for neuromuscular transmission. It is also one of the most complex channels in the pLGIC family, comprising of 4 different subunits - 2 alpha1, 1 beta1, 1 delta and either a gamma in foetal receptors, or an epsilon subunit in adult receptors. No structures of a full-length human muscle type nAChR receptor have been reported, with the closest homologue published being the electric organ nAChR of electric rays (Torpedo), which has a similar subunit composition to the foetal muscle nAChR. This proposal aims to obtain near atomic-resolution structures of full-length human muscle nAChR in multiple conformations, followed by targeted functional assessments of mutant channels to determine the structure-function relationship of the receptors.

Preliminary protein production and purification protocols have been established that enabled the collection of a small cryo-EM dataset from 5,911 particles of purified adult muscle nAChR. Views of the receptor from all angles were observed in 2D classes, giving a complete 3D reconstitution that produced an electron density map at 9 Å.

To obtain structures of the receptor in different conformations, we plan to trap it in separate functional states using established inhibitors such as alpha bungarotoxin and fluoxetine as well as a newly discovered positive allosteric modulator. To help obtain a structure of the open conformation, we will also try using slow-channel mutant receptors with an increased resting open probability.

After structures of the receptors have been elucidated, functional assessments of channels with mutations in key residues will be carried out using a combination of electrophysiology, biochemical and biophysical methods to see how these changes affect channel kinetics and affinity for different modulators.