Side Groups Convert the α7 Nicotinic Receptor Agonist Ether Quinuclidine into a Type I Positive Allosteric Modulator.

The quinuclidine scaffold has been extensively used for the development of nicotinic acetylcholine receptor (nAChR) agonists, with hydrophobic substituents at position 3 of the quinuclidine framework providing selectivity for α7 nAChRs. In this study, six new ligands (4-9) containing a 3-(pyridin-3-yloxy)quinuclidine moiety (ether quinuclidine) were synthesized to gain a better understanding of the structural-functional properties of ether quinuclidines. To evaluate the pharmacological activity of these ligands, two-electrode voltage-clamp and single-channel recordings were performed. Only ligand 4 activated α7 nAChR. Ligands 5 and 7 had no effects on α7 nAChR, but ligands 6, 8, and 9 potentiated the currents evoked by ACh. Ligand 6 was the most potent and efficacious of the potentiating ligands, with an estimated EC50 for potentiation of 12.6 ± 3.32 µM and a maximal potentiation of EC20 ACh responses of 850 ± 120%. Ligand 6 increased the maximal ACh responses without changing the kinetics of the current responses. At the single-channel level, the potentiation exerted by ligand 6 was evidenced in the low micromolar concentration range by the appearance of prolonged bursts of channel openings. Furthermore, computational studies revealed the preference of ligand 6 for an intersubunit site in the transmembrane domain and highlighted some putative key interactions that explain the different profiles of the synthesized ligands. Notably, Met276 in the 15' position of the transmembrane domain 2 almost abolished the effects of ligand 6 when mutated to Leu. We conclude that ligand 6 is a novel type I positive allosteric modulator (PAM-I) of α7 nAChR.

Regulation of nicotinic acetylcholine receptors by post-translational modifications.

Nicotinic acetylcholine receptors (nAChRs) comprise a family of pentameric ligand-gated ion channels widely distributed in the central and peripheric nervous system and in non-neuronal cells. nAChRs are involved in chemical synapses and are key actors in vital physiological processes throughout the animal kingdom. They mediate skeletal muscle contraction, autonomic responses, contribute to cognitive processes, and regulate behaviors. Dysregulation of nAChRs is associated with neurological, neurodegenerative, inflammatory and motor disorders. In spite of the great advances in the elucidation of nAChR structure and function, our knowledge about the impact of post-translational modifications (PTMs) on nAChR functional activity and cholinergic signaling has lagged behind. PTMs occur at different steps of protein life cycle, modulating in time and space protein folding, localization, function, and protein-protein interactions, and allow fine-tuned responses to changes in the environment. A large body of evidence demonstrates that PTMs regulate all levels of nAChR life cycle, with key roles in receptor expression, membrane stability and function. However, our knowledge is still limited, restricted to a few PTMs, and many important aspects remain largely unknown. There is thus a long way to go to decipher the association of aberrant PTMs with disorders of cholinergic signaling and to target PTM regulation for novel therapeutic interventions. In this review we provide a comprehensive overview of what is known about how different PTMs regulate nAChR.

Cannabidiol as a modulator of α7 nicotinic receptors.

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory 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 a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

Tyrosine phosphorylation differentially fine-tunes ionotropic and metabotropic responses of human α7 nicotinic acetylcholine receptor.

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.