The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-in ammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect, acting as a low-e cacy agonist and a non-competitive inhibitor. It activates α7 nAChRs, in line with our previous molecular dynamics simulations showing favorable binding of this accessible region of the S protein to the nAChR agonist binding site. However, activation requires the presence of positive allosteric modulators that enhance open probability, indicating very low e cacy. The main effect of the S fragment on α7 nAChR is a negative modulation, which is evidenced by a profound decrease in the durations of channel openings and activation episodes and in the amplitude of macroscopic responses elicited by ACh. Our study identi es a novel functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for exploring the involvement of nAChRs in COVID-19 pathophysiology.Research Centre (Grant Number:BB/L01386X/1), for funding ASFO and providing funds to purchase the Y674-R685 peptide from Designer Bioscience Ltd. (Cambridge UK).

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.