An experimental test of the nicotinic hypothesis of COVID-19.

The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a "nicotinic hypothesis," which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus' spike protein and small-molecule cholinergic ligands for the receptor's orthosteric binding sites. This notion is reminiscent of the well-established role of the muscle AChR during rabies virus infection. To address this hypothesis directly, we performed equilibrium-type ligand-binding competition assays using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-bungarotoxin (α-BgTx) as the orthosteric-site competing ligand. We tested different SARS-CoV-2 spike protein peptides, the S1 domain, and the entire S1-S2 ectodomain, and found that none of them appreciably outcompete [125I]-α-BgTx in a specific manner. Furthermore, patch-clamp recordings showed no clear effect of the S1 domain on α7-AChR-mediated currents. We conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR's orthosteric sites-and thus, its competition with ACh, choline, or nicotine-is unlikely to be a relevant aspect of this complex disease.

Probing function in ligand-gated ion channels without measuring ion transport.

Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel's affinities for ligands that bind to the extracellular domain.