Interpretation of Science Frontier Review︱Regulatory mechanism of nicotinic acetylcholine receptor accessory molecule and application prospects of disease treatment and transformation

Written by Sucre, edited by Wang Sizhen Acetylcholine (ACh) and its receptors play an extremely important role in the body's physiological functions and pathological processes
.

Physiologically, as an important neurotransmitter, ACh exerts corresponding physiological functions by interacting with different receptors.
For example, by interacting with nicotinic ACh receptors (nAChRs), it mediates a variety of physiological functions.
processes, including muscle contraction, nerve transmission, sensory transduction
.

In terms of pharmacology, nAChRs is a key mechanism of tobacco addiction, as well as an important therapeutic target for diseases such as hypertension and dementia [1]
.

 However, for a long time, due to our limited understanding of the mechanisms of nAChRs biosynthesis, folding assembly, transport process, and functional activation, and the hindered expression of functional nAChRs in recombinant cells, it has greatly hindered the basic research and drug development of nAChRs.
Process
.

Recently, the David S.
Bredt team from Johnson & Johnson Pharmaceuticals in the United States published the latest review article on Science titled Nicotinic acetylcholine receptor redux: Discovery of accessories opens therapeutic vistas
.

In this review, the author systematically reviewed the close coordination of various accessory protein molecules involved in the process of subunit synthesis, folding assembly, cell membrane transport and functional activation of various nicotinic acetylcholine receptors (nAChRs)
.

The discovery of these auxiliary molecules not only helps to strengthen our understanding of nAChRs biosynthesis, transportation, and biological functions, but also opens up new research fields and treatment directions for related diseases caused by abnormal nAChRs functions
.

 Background introduction 1.
The discovery of acetylcholine In 1921, Otto Loewi separated two beating frog hearts and placed them in Ren's solution.
The first heart was connected to the vagus nerve, and the other heart had the vagus nerve removed
.

Electrically stimulated the vagus nerve of the first heart, and found that the beating rate of the heart under its innervation decreased.
Then the Ren’s solution was sucked out and dripped into another heart with the vagus nerve removed.
As a result, it was found that the beating rate of the heart also decreased.
Therefore, Otto Leowi speculates that the nerves release special active substances through their ends to control the activity of the heart, and call the transferable substances "Vagusstoff" (Vagusstoff), which is the classic "double frog heart perfusion experiment
.
"
Then in 1929, Henry Dale and HW Dudley isolated from the spleens of bulls and horses and identified this active substance as acetylcholine [2-3]
.

Otto Leowi and Henry Dale jointly won the Nobel Prize in Physiology or Medicine in 1936 due to the above-mentioned pioneering work, which also opened the prelude to the study of acetylcholine
.

2.
Introduction to the biological functions mediated by acetylcholine receptors Acetylcholine functions by interacting with acetylcholine receptors
.

Acetylcholine receptors are divided into muscarinic Ach receptors (muscarinic Ach receptor) and nicotinic Ach receptors (nicotinic Ach receptor, nAChR).
The former is a G protein-coupled receptor and the latter is an ion channel receptor
.

Given that nAChR is the first neurotransmitter receptor isolated and also plays an important role in nerve conduction, in this review, the author focuses on nAChR
.

nAChR is widely distributed in tissues such as brain, muscle, lymphocytes, and cochlear hair cells, and regulates physiological functions such as cognition, muscle contraction, immune regulation, and auditory discrimination, respectively
.

 In addition, nAChR also plays an important role in a variety of pathological processes.
For example, the addictive effect of nicotine (nicotine) is closely related to the reward effect mediated by nAChRs [4]
.

Mutations in the nAChR subunit can lead to a variety of genetic diseases, such as epilepsy and neuromuscular junction disorders
.

nAChR polymorphism is associated with the occurrence of lung cancer
.

Therefore, in view of the important role of nAChRs in normal physiological functions and pathological processes, nAChR has gradually become an important target for the treatment and development of cardiovascular, mental and cognitive disorders [5]
.

3.
The structure of nicotinic acetylcholine receptors.
All nAChRs are similar in structure.
They are a pentameric structure composed of 5 subunits.
Each subunit contains an N-terminal extracellular domain for ligand binding, followed by Four transmembrane regions (transmembrane, TM)
.

Between TM3 and TM4 is a large cytoplasmic region composed of two structured helices MX and MA, which regulate the interaction with the cytoskeleton anchoring protein
.

The currently known subunits involved in the formation of nAChRs are: 9 α (α2-α10) subunits, 3 β (β2-β4) subunits, 1 γ subunit, 1 δ subunit, and 1 ε subunit
.

Among them, the nAChRs in neurons are mainly formed by a combination of various α2-α9 and β2-β4 subunits [6]
.

Research progress The biosynthesis of nAChRs in cells involves a highly complex and precisely regulated process
.

The role of a variety of molecular chaperones and accessory proteins in the processes of subunit synthesis, folding assembly, cell membrane transport and receptor function activation is the key factor for the normal physiological functions of nAChRs
.

1.
The nAChRs motor neurons in the muscle tissue are excited to release ACh, and the nAChRs acting on the skeletal muscles cause muscle excitement and contraction
.

However, the abnormal connection of ACh-nAChRs can lead to a variety of diseases
.

For example, mutations in genes involved in the formation of nAChRs are the main cause of congenital myasthenia syndrome; autoantibodies bind to proteins in neuromuscular junctions (acetylcholine receptors, muscle-specific receptors, titin, etc.
) to induce inflammation The reaction, which causes depletion of nAChRs, is an important mechanism leading to myasthenia gravis [7]
.

 At present, for myasthenia caused by decreased nAChR levels, cholinesterase inhibitors are mainly used to increase the level of acetylcholine in synapses
.

However, the curative effect of cholinesterase inhibitors is relatively mild, so there is an urgent need for better treatments
.

For example, replacing the mutated gene in patients with congenital myasthenia through gene therapy is a potential treatment method
.

In myasthenia gravis, although blocking the autoimmune process is an effective treatment, this method requires life-long immunosuppressive therapy
.

 Enhancing the assembly of nAChR and promoting the cell surface expression of nAChRs conceptually provides a new treatment strategy for some patients with myasthenia
.

People have a systematic knowledge and understanding of the conformational maturation and subunit oligomerization during the formation of nAChRs in muscle
.

Through forward genetic screening, CRELD1 protein was identified from Caenorhabditis elegans.
The study found that this molecule can promote the assembly and synthesis of nAChRs and is evolutionarily conservative.
Therefore, CRELD1 is a potential therapeutic target for myasthenia Click [8]
.

In addition, by enhancing the MuSK signaling pathway to promote nAChR aggregation in synapses, drugs aimed at treating neuromuscular connection disorders are currently under development
.

Figure 1 Auxiliary molecular screening process for functional receptor expression: functional biological experiment screening of proteins that can enhance receptor activity (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) 2.
NACHO as neuronal nAChRs The specific molecular chaperones express nAChRs in muscle in heterologous cell lines, which can form functionally active channels
.

The difference is that nAChRs in neurons are expressed in heterologous cell lines without functional activity
.

Through the method of genome-wide expression and cloning (Figure 1), it was discovered that a neuron-specific endoplasmic reticulum resident protein, NACHO, mediates the assembly of α7 nAChRs in neurons.
At the same time, this study also revealed that neuronal nAChRs are in Africa.
The reason why functional receptors cannot be formed in neuronal cells
.

NACHO mainly participates in the folding, assembly and membrane transport of the α7 subunit of α7 nAChRs through the cooperation of N-oligosaccharyltransferase (OST), calnexin, RIC3 and anti-apoptotic Bcl-2 protein [9]
.

In addition, NACHO is also involved in the biogenesis and function of other nAChRs, such as α4β2 nAChRs and α6β2β3 nAChRs
.

Subsequently, the study found that NACHO knockout mice showed hyperactivity and cognitive dysfunction, and this result was consistent with the phenotype caused by abnormal expression of nAChR, while the activity of other types of receptors, such as GABAA, 5- HT3A, AMPA, and TRPV1 behaved normally
.

Therefore, NACHO is a specific molecular chaperone of neuronal nAChRs (Figure 2)
.

Figure 2 The assembly, transport and activation mechanism of α7 nAChRs and α4β2 nAChRs (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) 3.
Regulation of nAChR assembly by drugs and metabolites Some small molecules can regulate nAChR Assembly
.

Based on the mechanism of action, these compounds can be divided into three categories
.

The first type is orthostructural ligands, which are mainly involved in the formation of Ach binding pockets in subunits and the stabilization of mature nAChRs
.

The second category includes menthol (menthol) and polyamines (polyamines), which can directly bind to the non-orthogonal binding site of nAChRs, thereby regulating the assembly of the receptor
.

The third category is some of the more common molecules involved in protein folding.
They generally do not directly act on AChRs.
They mainly include 4-phenylbutyrate, valproate, and butyrate.
And so on, they are also inhibitors of histone deacetylase
.

 In the 1980s, the first human study found that the level of nAChR in the brain of smokers increased [10]
.

Then, the study found that in animal experiments, nicotine can not only promote the function of nAChR, but also increase the post-transcriptional level of nAChR
.

This is completely different from some of our common G protein-coupled receptors (such as opioid receptors): after long-term exposure to agonists, the expression level will be down-regulated
.

Mechanistically, the interaction between nicotine and the ligand binding site promotes the formation of nAChR pentamers and the surface expression of the receptor, and these effects have nothing to do with the activation of the receptor
.

 In brain tissue, the strengthening properties of nicotine are mediated by nAChRs containing β2 subunits in the limbus of the brain.
Therefore, the up-regulation of nAChR is likely to be involved in nicotine addiction
.

Menthol can also increase the level of nAChRs in the brain
.

In transgenic mice, menthol can increase the assembly of nAChR containing α4 and α6 subunits in dopamine neurons [11]
.

Therefore, the increased expression of nAChRs in the reward loop may explain why menthol cigarette smokers are more difficult to quit than non-menthol cigarette smokers
.

 In addition, ligand-mediated assembly may also play an important role in the maturation of some nAChRs
.

The whole genome screened the factors that promote the normal function of α9α10 nAChRs, and identified the biosynthetic enzyme of acetylcholine, namely choline acetyltransferase (ChAT)
.

ChAT-mediated assembly of α9α10 subunits reflects the molecular chaperone effect of Ach [12]
.

Since Ach cannot effectively penetrate the cell membrane, it is implied that Ach may promote the assembly of α9α10 nAChR through extracellular mechanisms
.

In fact, even if α-cyclosnake toxin is applied outside the cell (which cannot penetrate the cell membrane), it can also increase the level of α9α10
.

Therefore, this assembly mechanism mediated by Ach extracellularly may contribute to the positioning of the functional α9α10 subunit receptor at the Ach release site.

.

 With the help of cDNA expression cloning technology (Figure 3), the molecule SAT1 that enhances the surface transport of α4β2 was identified
.

SAT1 is the rate-limiting enzyme of polyamine catabolism, suggesting that these ubiquitous linear polymeric cations can inhibit the expression of α4β2
.

Indeed, blocking the synthesis of polyamines can increase the level of functional α4β2 subunit and α7 subunit receptors
.

In addition, SAT1 gene polymorphism has a strong correlation with suicidal behavior
.

Therefore, these research results provide new evidence for further evaluation of nicotine preparations as drugs for treating suicidal behavior
.

Figure 3 Auxiliary molecular screening process for functional receptor expression: high internal content imaging technology to screen proteins that can promote receptor transport (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) 4.
Auxiliary protein pair Regulatory role of nAChR function The main function of protein chaperones is to help the assembly and transport of nAChR receptors, while auxiliary molecules are mainly to stabilize receptors and control channel functions
.

In C.
elegans, two nAChR accessory molecules have been reported successively, MOLO-1 and EAT-1, which are involved in the functional activation and functional expression of nAChRs respectively, and both are single transmembrane proteins [13-14]
.

 Similarly, the biosynthesis of a variety of mammalian nAChRs also involves accessory molecules
.

Studies have found that the assembly, transport and functional activation of α6β2β3 nAChR receptors in the striatum involve the participation of a variety of accessory molecules
.

α6β2β3 nAChRs is a presynaptic receptor sensitive to conotoxin MII
.

However, in the striatum of NACHO knockout mice, the conotoxin MII receptor binding site is reduced
.

In addition, in the HEK293 cell line, NACHO co-transfection can promote the assembly of oligomers, but cannot express the functional α6β2β3 nAChR receptor
.

Furthermore, using the whole genome screening method, it was identified that SULT2B1, LAMP5 and BARP cooperate with NACHO to reconstruct the function of α6β2β3 nAChR channel protein
.

Specifically, NACHO promotes the assembly of α6β2β3 nAChRs, LAMP5 and SULT2B1 promote the cell surface transport of receptors, and BARP enhances the activity of receptor channels [15] (Figure 4)
.

Figure 4 The regulatory mechanism of the midbrain dopamine neuron α6β2β3 nAChRs function (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) The functional reorganization of different nAChRs involves the participation of different accessory proteins
.

α6β4 nAChRs are mainly distributed in the sensory neurons of the dorsal root ganglia and have important potential pharmacological values
.

Different from α6β2β3 nAChRs, the functional recombination of α6β4 nAChRs requires the participation of BARP and IRE1α accessory molecules, and is not affected by NACHO [16] (Figure 5)
.

IRE1α is a sensor that responds to unfolded proteins
.

The α6β4 subunit is a potential therapeutic target for neuropathic pain
.

Figure 5 The regulatory mechanism of α6β4 nAChRs function in dorsal root ganglion sensory neurons (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) Similarly, the functional assembly of α9α10 nAChR also requires specific accessory proteins to participate
.

Through the method of genome-wide expression and cloning, TMIE and TMEM132E were identified as important auxiliary molecules for the functional assembly of α9α10 nAChR
.

Both TMIE and TMEM132E are single-pass transmembrane proteins, and both have recessive mutations in non-syndromic deafness [17-18] (Figure 6)
.

The above-mentioned studies indicate the diversification of the function regulation of nAChRs
.

Figure 6 Cochlear hair cell α9α10 nAChR assembly (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) In addition, many strong affinity toxins (prototoxin) can bind stably with different nAChRs, thereby affecting the corresponding nAChRs Physiological function
.

For example, the strong affinity toxin lynx1 affects the assembly of α4β2 nAChRs [19], and lynx2 inhibits the physiological functions of α4β2 nAChRs and α7 nAChRs [20].
The strong affinity toxin targets the diversified effects of nAChRs, and is used for clinical development of mental, pain and other diseases.
Therapeutic intervention provides new possibilities and directions
.

In addition, how the strong affinity toxins functionally interact with nAChR receptor accessory molecules, such as NACHO and 14-3-3, is also a very interesting research area in the future
.

  5.
The influence of auxiliary components on the neurobiology of nAChR In combination with the above content, multiple molecules and signal pathways are involved in the functional regulation of nAChRs
.

Some are small molecules, and some are small protein molecules.
They are either involved in regulating the biosynthesis of receptors or in regulating the activation of channels
.

It is worth noting that different types of nAChRs are selective for auxiliary molecules.
For example, NACHO and BARP can regulate a variety of nAChRs, while TMIE, TMEM132e and IRE1a can only affect a single nAChR type.

.

At the same time, the functional expression of some nAChRs requires a variety of accessory molecules (Table 1)
.

This selectivity and complexity, on the one hand, reflects that the orderly expression of nAChRs in specific tissue types and cell specific locations requires precise coordination between different molecules, and on the other hand, it also lays the foundation for the extensive physiological functions of nAChRs in the body.
Molecular basis
.

Table 1 Auxiliary molecules involved in regulating the biosynthesis and function of nAChRs (Table quoted from: Matta JA, Gu S, et al.
, Science.
2021) 6.
The influence of auxiliary components on the neuropharmacology of nAChR to realize the functional expression of nAChRs as The development of drugs for various diseases, such as chronic pain, Parkinson's disease and hearing impairment, offers the possibility
.

Studies have found that epibatidine, an alkaloid derived from frog skin, is a ubiquitous nAChR agonist and is currently a powerful analgesic [21]
.

In addition, a pan-nAChR agonist, ABT-594, has a very good effect on diabetic nephropathy.
It is currently in phase II clinical trials.
However, its mechanism of targeting nAChR is still unclear
.

 Subsequently, genomic screening of unrelated mouse dorsal root ganglia found that the mRNA level of nAChR α6 subunit was negatively correlated with the pain response in the non-neural injury mouse model
.

In addition, the study found that the analgesic effects of nicotine in mice with α6 knockout disappeared after inflammation and neuropathic injury
.

Another study found that human postoperative pain and temporomandibular joint disorder syndrome are affected by CHRNA6 (α6) promoter polymorphism
.

Therefore, α6β4 may be a potential target for the treatment of chronic pain
.

The functional recombinant expression of human α6β4 through BARP and IRE1α has paved the way for the development of drugs against this target [22]
.

 The other two nAChRs with potential therapeutic value are α6β2β3 nAChR and α9β10 nAChR
.

Thanks to their smooth expression in mammalian cells, it has greatly promoted the research on the biochemical properties of the corresponding receptors and the high-throughput drug screening and development
.

 In the brain, α6β2β3 nAChR receptors are abundantly expressed in the presynaptic nerve terminals of neurons in the limbic system of the brain.
At the same time, in the striatum and nucleus accumbens, α6β2β3 nAChRs mediate the release of dopamine induced by acetylcholine and nicotine
.

Therefore, this makes α6β2β3 nAChRs a potential target for the treatment of Parkinson's disease, schizophrenia and drug addiction
.

 α9β10 nAChR is abundantly expressed in the outer ear hair cells of the adult cochlea and mediates the transmission of information from the superior olivary complex
.

Incoming signals reduce the amplification of outer ear hair cells through a negative feedback mechanism.
This process effectively improves the signal resolution ability in background noise, while also protecting hearing from sound damage [23]
.

In addition, studies have found that the abnormality of the negative feedback mechanism is likely to be related to the pathogenesis of tinnitus
.

Therefore, the functional expression of α9β10 nAChR will not only open up a new path for drug development, but also provide a new direction for the treatment of hearing disorders
.

 Some other important nAChRs, such as nAChRs containing α2 or α5 subunits, are still difficult to express in recombinant cell lines and lack selective drugs, which has brought many difficulties for in-depth research
.

α2β2 nAChR receptors are expressed in hippocampal OLM (oriens lacunosum-moleculare) neurons and participate in the synaptic plasticity caused by nicotine.
Therefore, α2β2 nAChR is a potential target for the treatment of cognitive dysfunction [24]
.

It is worth noting that among all human single nucleotide polymorphisms, the genetic polymorphism of the α5 nAChR subunit (CHRNA5) is second only to ApoE4 in determining human lifespan
.

 Summary and prospects ACh-nAChRs play an important role in the physiological and pathological processes of the body
.

nAChRs are widely distributed in tissues such as brain, muscle, lymphocytes and cochlear hair cells, and mediate physiological functions such as cognition, muscle contraction, immune regulation, and auditory discrimination, respectively
.

In addition, nAChRs also play an important role in deafness, addiction, cancer, mental illness, cardiovascular, hypertension and other diseases (Figure 7)
.

Figure 7 The tissue distribution, physiological function and biosynthesis of nAChRs (picture quoted from: Matta JA, Gu S, et al.
, Science.
2021) If we say that the discovery of "vagus neuron" (acetylcholine) and nAChRs opens up our understanding of ACh -The gate of nAChRs understanding, so the discovery of auxiliary molecules represents the beginning of a turning point in our understanding of nAChRs biology
.

 The types of nAChRs are diverse and the composition is complex.
Different types of nAChRs have different subunits.
The assembly or activation of certain types of nAChRs requires specific accessory proteins to participate, and certain accessory proteins can participate in the synthesis of multiple nAChRs, such as NACHO And BARP can regulate a variety of nAChRs, while TMIE, TMEM132e and IRE1a can only affect a single nAChR type
.

In addition, there are still many important nAChRs that are difficult to express in recombinant cell lines and lack selective drugs, which brings many difficulties for in-depth research on related receptors
.

With the discovery of auxiliary molecules in the process of nAChRs biosynthesis, we have gradually deepened our understanding of the process of nAChRs biosynthesis, specifically, the synthesis, folding assembly, membrane transport and function of a variety of small molecules or proteins in the subunits of nAChRs Play a regulatory role in the activation process
.

The in-depth study of this process provides more therapeutic targets that can be intervened for the research of related diseases
.

The complexity of nAChRs biosynthesis, transport, and biological functions, as well as the fine regulation of these processes by nAChRs accessory molecules, have jointly laid the molecular basis for nAChRs and accessory molecules to participate in the body's physiological, pathological, and pharmacological processes
.

At the same time, they also jointly provide favorable conditions for the development of specific disease interventions, including mental diseases, neurological diseases, and hearing disorders
.

 Original link: https:// Selected articles from previous issues [1] JAMA Neurol︱ Attention! Young people are more likely to suffer from "Alzheimer's disease"? [2] Cereb Cortex︱ oxytocin can regulate the individualized processing of facial identities and the classification of facial races in the early facial regions of the brain [3] Nat Commun | Qi Xin's research group revealed that the compound CHIR99021 can treat Huntington's dance by regulating mitochondrial function The molecular mechanism of the disease [4] Cereb Cortex︱ Ku Yixuan’s team reveals the ipsilateral sensory cortex representation pattern of working memory [5] Neurosci Bull︱ synapse-associated protein Dlg1 improves depression-like behavior in mice by inhibiting microglia activation [6 】Brain | For the first time! PAX6 may be a key factor in the pathogenesis of Alzheimer's disease and a new therapeutic target [7] Sci Adv︱ blockbuster! DNA methylation protein DNMT1 mutation can induce neurodegenerative diseases [8] Cell︱ new discovery! New enlightenment of midbrain-regulated movement phenomenon for the treatment of Parkinson’s disease [9] Cereb Cortex︱MET tyrosine kinase signal transduction timing abnormality is a key mechanism affecting the development and behavior of normal cortical neural circuits in mice [10] Nat Biomed Eng︱ The team of academician Ye Yuru develops a new strategy for whole-brain gene editing-mediated treatment of Alzheimer’s disease [11] Luo Liqun Science's heavy review System interpretation ︱ Neural circuit structure-a system that makes the brain "computer" run at high speed [12] Sci Adv ︱Important discovery! The calcium homeostasis regulatory protein Calhm2 regulates the activation of microglia and participates in the process of Alzheimer's disease [13] EMBO J︱ new discovery! AGHGAP11B promotes the expansion of the neocortex into adulthood and improves cognitive ability [14] Cell Death Differ︱ Qi Yitao/Wu Hongmei and others cooperate to reveal the molecular mechanism of SUMO modification regulating neurogenesis in adult mice [15] Cereb Cortex︱A2A receptor antagonist can Reversal of sequence learning impairment induced by abnormal aggregation of α-Syn References (slide up and down to view) 【1】X.
Zhang et al.
, Brain control of humoral immune responses amenable to behavioural modulation.