Radial Tilting of the Extracellular Domain of GLIC Revealed by EPR Spectroscopy

Abstract

Signaling in the brain depends on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). Defects in these proteins are linked to a variety of diseases including myasthenia, epilepsy and sleep-disorders. In addition, these proteins are therapeutic targets for various drugs including anti-convulsants, anxiolytics, anesthetics and sedatives. While recent crystal structures of prokaryotic and eukaryotic pLGICs solved in detergent micelles have shed light on the molecular architecture of these proteins, describing their conformational dynamics in physiological lipids is essential for understanding their function. Crystal structures of GLIC and the glutamate-activated chloride channel predict that agonist-mediated channel opening is associated with a quaternary twisting and a radial un-blooming (inward tilting) of the extracellular binding domain (ECD). Using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy and functional GLIC channels reconstituted in liposomes, we are testing these gating predictions. Residues located at the top of β-strands (β1, 2, 5, 6, 8) were mutated to cysteine, labeled with the spin probe MTSL and proton-induced gating motions were measured using continuous wave (CW) and double electron-electron resonance (DEER) spectroscopy. CW EPR spectroscopy data showed the spin label attached to I92C, S93C, V94C (β5), T99C and Q101C (β6) moved into a slightly more constrained/immobile environment at pH 3.0 as compared to pH 7.6. DEER distance distributions for K48 (β2), S93 (β5), T99 and Q101 (β6) revealed the probes moved closer together at pH 3.0, suggesting that the inner β-sheet of the extracellular domain of GLIC tilt inwards upon agonist activation.