Structural Requirements in the Transmembrane Domain of GLIC Revealed by Incorporation of Noncanonical Histidine Analogs

• Published in: Chemistry & biology Vol. 21; no. 12; pp. 1700 - 1706
• Main Authors: Rienzo, Matthew, Lummis, Sarah C.R., Dougherty, Dennis A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 18-12-2014
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cell Membrane - metabolism; Cyanobacteria; Histidine - analogs & derivatives; Hydrogen Bonding; Hydroxy Acids - chemistry; Ligand-Gated Ion Channels - chemistry; Ligand-Gated Ion Channels - genetics; Ligand-Gated Ion Channels - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Protein Structure, Tertiary
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/j.chembiol.2014.10.019

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using in vivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism.
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•A backbone mutation in M2 reveals the importance of an interhelix hydrogen bond•Two noncanonical histidine analogs synthesize and incorporate into GLIC•His234 is a sterically restricted proton-binding site essential for gating
Rienzo et al. examine a role of a key histidine residue, His234, in the prokaryotic ion channel GLIC by introducing noncanonical amino acids. This allows them to probe specific features, such as a critical interhelix hydrogen bond, and provides support for the proposed role of His234 in channel gating.