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Edited by Jean-Pierre Changeux, CNRS, Institut Pasteur, Paris, France, and approved January 21, 2015 (received for review September 16, 2014)
Significance
Pentameric ligand-gated ion channels (pLGICs) mediate neuronal communication in the central nervous system. Upon the neurotransmitter binding, these receptors undergo a rapid conformational change to open an integral ion channel. Mutations impairing the function of pLGICs are known to cause hyperekplexic, myasthenic, and epileptic syndromes. Here, we studied how the local perturbations caused by single mutations result in an alteration of the protein function. Using a chimeric protein assembled by the transmembrane domain of the human glycine receptors fused to the extracellular domain of the bacterial pLGIC GLIC, we performed functional experiments in parallel with X-ray crystallography. On this basis, we propose a molecular mechanism for channel opening that accounts for the phenotypes of several mutants causing hyperekplexia.
Abstract
The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.
Footnotes
↵1G.M.-C., L.S., and C.H. contributed equally to this work.
- ↵2To whom correspondence may be addressed. Email: pjcorrin{at}pasteur.fr or delarue{at}pasteur.fr.
Author contributions: G.M.-C., L.S., M.D., and P.-J.C. designed research; G.M.-C., L.S., C.H., L.M., C.G.-B., S.P., and S.M. performed research; A.T. and M.B. contributed new reagents/analytic tools; G.M.-C., L.S., C.H., L.M., S.M., A.T., M.B., M.D., and P.-J.C. analyzed data; and G.M.-C., L.S., M.D., and P.-J.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4X5T).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1417864112/-/DCSupplemental.
X-ray structure of the TMD of the α1GlyR
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