Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-Gated Ion Channel (GLIC) Supports Conserved Gating Motions Among Cys-Loop Receptors

Abstract

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro119 in the Cys-loop, Pro198 and Pro203 in the M1 helix, and Pro299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro119, Pro203, and Pro299 mutants are consistent with earlier proline mutagenesis work. However, the Pro198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

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