Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors.


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Authors
Rienzo, Matthew 
Rocchi, Angela R 
Threatt, Stephanie D 
Dougherty, Dennis A 
Lummis, Sarah CR 
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. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 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 Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

Description
Keywords
Cys-loop receptor, Gloeobacter violaceus, electrophysiology, mutagenesis, non-canonical amino acid, non-standard mutagenesis, nonsense suppression, proline analogs, protein conformation, structure-function, Amino Acid Motifs, Amino Acid Sequence, Amino Acid Substitution, Bacterial Proteins, Cyanobacteria, Hydrogen Bonding, Ion Channel Gating, Ligand-Gated Ion Channels, Models, Molecular, Molecular Sequence Data, Proline, Protein Structure, Secondary
Journal Title
J Biol Chem
Conference Name
Journal ISSN
0021-9258
1083-351X
Volume Title
291
Publisher
Elsevier BV
Sponsorship
This work was supported by grants from the NIH (NS 34407 to DAD) and the Wellcome Trust (WT 81925 to SCRL), and an NIH/NRSA training grant (GM07616 to MR).