Identification of a Cannabidiol Binding Domain Involved in the Allosteric Modulation of the alpha7 Nicotinic Acetylcholine Receptor

Abstract

The α7 nicotinic acetylcholine receptor is a homopentameric ligand-gated ion channel widely expressed in the brain and also present in non-neuronal cells. It contributes to cognition, memory, neuroprotection, and the control of inflammation. Reduced α7 activity has been associated with neurological and neurodegenerative disorders, whereas enhanced activity may promote cancer progression. Thus, both potentiation and inhibition of α7 are of therapeutic interest. Cannabidiol (CBD), a non-psychoactive compound with therapeutic potential and broad use, is an α7 modulator. Using patch- clamp recordings, we found that CBD rapidly decreases the frequency of α7 activation episodes while also inducing delayed, prolonged openings, suggesting multiple binding sites or mechanisms. To predict binding sites, we performed coarse-grained molecular dynamics simulations of α7 in resting and desensitized states embedded in a POPC:POPA:CHOL membrane in the presence of CBD. Several potential sites were identified. Representative structures were backmapped to atomistic detail and simulated to analyze stability and interactions. In the desensitized state, two residues from M4 interacted with CBD, while two others from M1 contributed to the resting site. Furthermore, one residue from the Cys-loop was involved in both states. These residues define a putative CBD binding domain located at the extracellular-facing apex of the M1 and M4 helices. To test their functional contribution, residues were mutated to alanine and electrophysiological recordings were performed. Mutants displayed altered responses to CBD: some mutations primarily reduced sensitivity to inhibition by affecting opening frequency, whereas others prolonged activation episodes by modifying channel kinetics. These effects did not segregate strictly by site, as residues from different locations showed overlapping functional roles. Taken together, our results support the existence of a CBD binding domain that allosterically modulates α7. Understanding these mechanisms is crucial for the rational therapeutic use of CBD and for anticipating possible adverse effects.