venoms and brain acetylcholine receptors

What is the nicotinic acetylcholine receptor?

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. The receptor has three states: resting, active, and desensitized. This image was created with information from rcsb.org, The top view shows the Ca²⁺ channel going into the cell. Note that this channel is a pentomer, composed of five sub units. Two versions of the side view are shown: one of the pentomer and one of just one of five sub units. The binding site for an inhibitor is expanded upon in the bottom image to show the ligand binding.

What are some ligands of interest?

The nicotinic acetylcholine receptor is named after nicotine, not to be confused with nicotinic acid. Nicotine is an agonist of nAChR. Epibatine is a chlorinated alkyloid from poison frogs of Ecuador. Epibatidine causes paralysis and analgesia. Cuprous nicotinic acid is a similar compound of interest.

Proceeding to nAChR, Covid, and snake venom..

This post is going to briefly acknowledge that the SARS-CoV2 virus binds to neurophilin 1 in addition to the ACE2 receptor. [1]

The following is a table compiled from Table 1 (just conotoxins) and Table 2 (other snake peptide toxins) from reference [2].

toxin	snake	AChR isoform	IC50
Conotoxin ImI	C.imperialis	hα7	595 nM
α-bungarotoxin	Multibanded krait	hα7	0.4 nM
α-cobratoxin	Indo Chinese spitting cobra	hα7	0.3 nM
αδ-bungarotoxin-1	Blue krait	hα7	1.18 nM
Drysdalin	White lipped snake	hα7	10 nM
Haditoxin	King cobra	hα7	10uM

A relevant point is that some of these venoms have extremely high affinity for the human α7 isoform of the neuronal acetylcholine receptor. (hα7). This post will present arguments that regions of the pike protein have limited homology to high affinity snake venoms. To put things into perspective for lay readers,

• a mole is 6.02×10²³ ( 602,000,000,000,000,000,000,000 ) molecules.
• 1 nM is 0.000000000001 moles per liter, or 602,000,000,000,000 molecules per liter. To achieve 50% of the maximum inhibition (IC₅₀) in a tiny 10 microliter drop, one needs 6,020,000,000 molecules

Mapping nicotinic acetylcholine receptors in the brain.

Judging by Proteinatlas.org brain mRNA map, neurophilin is expressed mostly in the cerebral cortex and hippocampus formation. ACE2 is practically undetectable in the brain. This post addresses the popular press frenzy that Covid treatments contain venom toxins. These are some brain maps of sub units of two of the three nicotinic acetylcholine receptors found in the brain. [2]

CHRNA7 is the alpha sub unit of the homo tetramer form of the neuronal aetylcholine receptor that this post is most interested in because it binds to the Coivid spike protein. [3,4]. This isoform expression is most expressed in components of the brain stem.

The locus ceruleus of the brain stem might be of interest in some who think that copper deficiency may be part of Long Covid. The locus ceruleus contains a enzyme called dopamine hydroxylase that synthesizes norepinephrine from dopamine. These neurons receive colinergic input. nAChR isoforms in the rat brain have been mapped by Léna et al 1999. The β2 subunit was present in all neurons, and the β3, α6, and α4 subunits were present in more than 50% of the neurons sampled. The subunits α3, β4, and α5 were found in less than 50% of the sample. α7 was found in only seven neurons (20%) and α2 was found in a single neuron. The authors also discussed the tendency of a particular subunit to be found with other subunits.

The Covid and Cigarette smoking paradox

Back in 2020 it was noted that cigarette smokers were under represented in the Covid-19 intensive care patients. [3] These patients have lung damage from any number of components in cigarette smoke. Why are they not over represented? This observation prompted Farsalins and colleagues to test the hypothesis that the Covid spike glycoprotein has sequence homology to any number of the sequences of the snake venom toxins that bind to the many forms of the nicotinic acetylcholine receptors. [3] Farsalino and coauthors found some weak homology between part of the spike glycoprotein and the Chinese cobra, Naja atra, neurotoxin homolog NL1. [3] Farsalinos and coauthors published some images of their in silico molecular docking but did not publish the apparent affinities.

Lagoumintzis and others from the same group published in silico estimated affinities in 2021 [4] In this case they used the original SARS-Cov spike protein and he Covid-19 SARS0CoV-2.

In extremely simple terms, the more negative Gibbs free energy (ΔG) is, the more spontaneous the binding reaction is. The smaller the dissociation constant (Kd) the higher the affinity. 4.6e-08 is smaller than 1.3E-07 indicating that the Covid-19 spike protein has a 10x higher affinity for the open than the closed alpha7 nAChR. Lysine K214 and phenylalanine F209 are predicted to be key residues in α7. [4] Electrostatic energy has to do with positive/negative charge interactions. Buried surface area is that surface not exposed to the solvent, generally hydrophobic amino acids.

Another SARS-CoV2 spike protein cryptic snake venom site

Proceeding to another similar in silico study by Oliveira and coworkers. This time the spike protein region was Tyrosine 674 to Arginine 685. The authors were comparing the sequence to a rabies virus toxin and α-bungarotoxin. In the torpedo muscle like receptor αβγδ receptor, the two binding pockets are nonequivalent; one is formed by an α and a δ and the second by α- and γ-subunits.

Here is another interesting figure comparing the binding sites. The Coivd spike protein segment is in deep magenta.

Text from the publication [4] were extracted and put into table form Interacting pairs are the same color. For instancetyrosine YY223 of nAChR subunit α4 hydrogen bonds with glutamine Q675 of the spike protein. Likewise the nAChR subunit β2 has a glutamate D195 amino acid that forms a hydrogen bond with alanine A684 of the spike protein.

nAChR	AChR	spike	AChR	spike	AChR	spike
Hydrogen bonds
α4β2	a4 Y223	Q675	b2 S192	N679	b2 D195	A684
α7	a7 D186	Q675	a7 Y210	Q677
αβγδ	a Y214	Q675	d D186	N679
Arginine 682 interacting with  tyrosine aromatic ring electrons.
α4β2	a4 Y223	R682	a4 Y230,	R682	a4 Y214	R682
α7	a7 Y115	R682	a7 Y217	R682	a7 Y210	R682
αβγδ	a Y117	R682	aY222	R682

Behavior observed for the peptides in the two binding pockets:

 When bound to the α4β2 and α7 nAChR, the peptide adopted many different binding modes inside the pocket, ranging from highly compact to fully extended conformations.  The torpedo muscle subtype was more compact with limited dynamical freedom.   At the start of simulations the spike protein peptide adopted an open conformation.  The peptide moved deeper into the pocket of the α7 AChR in a manner that might be associated with agonist opening of the channel.

Does gycosylation of the spike glycoprotein shield the Y674-R685 like loop from binding to the nAChR?  The simulations suggest that this is not the case.  

Blue, the receptor binding (ACE2) motif of the SARS-CoV2 spike protein.  Recall the fist SARS-CoV venom motif was (aa 362–377) [4] The SARS-CoV-2 RBM (aa 375–390) is close. [4] Yellow, glycosylation.  Middle, The spike protein (bottom view)  binding to the nAChR (side view).  Right, A top view of nAChR and a side view of the spike protein.   What seems to be missing is whether there is steric hindrance between the virus capsid and the cell membrane of the host. 

Concluding thoughts

In summary, we have two accounts of snake venom motifs in the SARS-CoV2 spike protein that are predicted to bind to one or more of the neuronal nicotinic acetylcholine receptors.  The Italian investigators found the venom motif at the top of the spike putting it really close to the ACE2 binding motif.  The British group found a motif on the side of the spike.  The first group predicted an affinity that was substantially less than the impressive nM affinity of select snake venom for nicotinic acetylcholine receptor family members.  The second group came up with some really impressive hypotheses on how binding the spike protein to the Ca²⁺ channel would cause closing or opening of the channel.    As exciting as these in silico research studies might be, there are some other realities that cannot be addressed with software.

The spike protein is attached to the rest of the virus. 

1. In the side binding model, would the virus press into the body of the cell?  How much this tend to mechanically dislodge the binding at nAChR? 
2. In either model, could the presence of the rest of the virus present mechanical torque that could open or close the channel in unexpected ways?
3. One virion could theoretically bind to multiple nAChR on one cell or bridge adjacent cells together.  Could these simultaneous interactions exert mechanical influences on the cell f in which these nAChR ion channels are dissolved? 

More questions

1. Can binding to nAChR result in SARS-CoV2 viral entry?
2. If the virions come from adjacent astrocytes, does the virus just stop at inhibiting and/or opening nAChR Ca²⁺ channels?
3. Normally astrocytes remove neurotransmitters from the synaptic cleft.  How quickly is the SARS-CoV2 virus cleared? What does it mean for a potential agonist or antagonist sticking around?

Let’s just do the experiment!

We obviously are not going to viable SARS-CoV2 virus! The Naval Research Laboratory and the National Center for Advancing Translational Sciences have developed a Quantum Dot system that is conjugated with the Covid spike protein. [8] In very simple terms, QDs are super fluorophores. These authors expressed the ACE2 receptor as a chimera with a yellow fluorescent protein in a cell culture system. [6] The yellow fluorescent protein absorbs light in the green range of 435–550 nm and emits yellow orange light that is absorbed by the QD The QD emit 608 nm red light.

This system not only allowed the investigators to follow the uptake of fake virus but also proved that the virus was using the ACE2 receptor because fluorescence resonance energy transfer requires objects to be fairly close. This assay would probably work just by monitoring uptake of the QD into cells expressing the nicotinic acetylcholine receptor. This Gorshkov study used neutralizing antibodies to inhibit endocytosis. Inhibitory small molecules could also be investigated.

References

1. Gadanec, L. K., McSweeney, K. R., Qaradakhi, T., Ali, B., Zulli, A., & Apostolopoulos, V. (2021). Can SARS-CoV-2 Virus Use Multiple Receptors to Enter Host Cells?. International journal of molecular sciences, 22(3), 992. free article
2. Bekbossynova, A., Zharylgap, A., & Filchakova, O. (2021). Venom-Derived Neurotoxins Targeting Nicotinic Acetylcholine Receptors. Molecules (Basel, Switzerland), 26(11), 3373. free article
3. Farsalinos, K., Eliopoulos, E., Leonidas, D. D., Papadopoulos, G. E., Tzartos, S., & Poulas, K. (2020). Nicotinic Cholinergic System and COVID-19: In Silico Identification of an Interaction between SARS-CoV-2 and Nicotinic Receptors with Potential Therapeutic Targeting Implications. International journal of molecular sciences, 21(16), 5807. https://doi.org/10.3390/ijms21165807 PMC free article
4. Lagoumintzis, G., Chasapis, C. T., Alexandris, N., Kouretas, D., Tzartos, S., Eliopoulos, E., Farsalinos, K., & Poulas, K. (2021). Nicotinic cholinergic system and COVID-19: In silico identification of interactions between α7 nicotinic acetylcholine receptor and the cryptic epitopes of SARS-Co-V and SARS-CoV-2 Spike glycoproteins. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 149, 112009. PMC free article
5. Oliveira, A., Ibarra, A. A., Bermudez, I., Casalino, L., Gaieb, Z., Shoemark, D. K., Gallagher, T., Sessions, R. B., Amaro, R. E., & Mulholland, A. J. (2021). A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors. Biophysical journal, 120(6), 983–993. https://doi.org/10.1016/j.bpj.2021.01.037 PMC free article
6. Gorshkov, K., Susumu, K., Chen, J., Xu, M., Pradhan, M., Zhu, W., Hu, X., Breger, J. C., Wolak, M., & Oh, E. (2020). Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis. ACS nano, 14(9), 12234–12247. PMC free article