Vestibular Modulation by Stimulant Derivatives in a Pentameric Ligand-Gated Ion Channel

Allosteric modulation of pentameric ligand-gated ion channels (pLGICs) is critical to the action of neurotransmitters and many psychoactive drugs. However, details of their modulatory mechanisms remain unclear, especially beyond the orthosteric neurotransmitter-binding sites. The recently reported prokaryotic channel sTeLIC, a pH-gated homolog of eukaryotic receptors in the pLGIC family, is thought to be modulated by aromatic compounds via a relatively uncharacterized modulatory site in the extracellular vestibule. Here, we show that sTeLIC is sensitive to potentiation by psychostimulant derivatives. By determining new cryo-EM and X-ray structures in closed and open states, and testing the impact of targeted mutations on electrophysiological behavior, we show that several amphiphilic compounds preferentially bind a vestibular pocket in the contracted open-state extracellular domain. This work provides a detailed structure-function mechanism for allosteric potentiation via a noncanonical lig- and site, with potential conservation in eukaryotic pentameric ligand-gated ion channels. ### Competing Interest Statement The authors have declared no competing interest. * 5-HT3 A R : Serotonin-3A receptor BrAmp : 4-Bromoamphetamine BrCA : 4-Bromocinnamate BrPEA : 4-Bromophenethylamine Cryo-EM : Cryogenic electron microscopy EC50 : Half-maximal effective concentration ECD : Extracellular domain FFC-8 : Fluorinated fos-choline-8 ICD : Intracellular domain pLGIC : Pentameric ligand-gated ion channel SEM : Standard error of mean TEVC : Two-electrode voltage-clamp TMD : Transmembrane domain WT : Wild type