Abstract
Neurosteroids are endogenous sterols that modulate pentameric ligand gated ion channels (pLGICs), and can be anesthetics, analgesics, and anti-epileptics. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. However, the structural determinants of these sites remain unknown. We used a novel series of neurosteroid photolabeling reagents combined with top-down and middle-down mass spectrometry to determine the stoichiometry, sites and orientation of neurosteroid binding in Gloeobacter ligand gated ion channel (GLIC), a prototypic pLGIC. Neurosteroids photolabel two sites per GLIC subunit, both within the transmembrane domain; one site is an intrasubunit site and the other is located in the interface between subunits. By using complementary photo-reactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Docking simulations are consistent with both sites being neurosteroid binding sites. Mutations introduced to either site alter neurosteroid modulation of GLIC channel activity demonstrating the functional role of both sites. These results provide a detailed molecular framework for understanding multi-site allosteric modulation of the family of pLGICs by neurosteroids.
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call