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.
Highlights
Nels, all of which share a pentameric structure and a similar global layout
Conventional Mutagenesis Identifies Four Sensitive Proline Sites—GLIC contains 18 proline residues, 12 of which are present in related receptors (Fig. 1)
Examination of the available GLIC structural data reveals that the conformational effects conferred by these residues are consistent with those of previously studied prolines with the same phenotypes: Pro-119 is located in a potentially flexible loop; Pro-299 forms a kink in M4 due to simple ablation of a hydrogen bond; and Pro-203 exists below a prominent bulge and displays steric requirements we have previously reported for a conserved bulge-inducing proline in the D2 dopamine receptor [32]
Summary
Molecular Biology—The cDNA for GLIC was in the pGEMhe plasmid. Site-directed mutagenesis was performed using the Stratagene QuikChange protocol to generate the appropriate codon. Each cell was injected with 50 –100 ng each of receptor mRNA and appropriate tRNA ϳ48 h before recording. For wild type experiments and conventional mutants, each cell received a single injection of 1–25 ng of receptor mRNA ϳ24 h before recording. As a negative control for suppression experiments at each site, unacylated full-length tRNA was co-injected with mRNA in the same manner as charged tRNA. These experiments yielded negligible responses for all sites. Wild type recovery conditions (injecting tRNA charged with the appropriate amino acid to regenerate a wild type channel via nonsense suppression at a TAG stop codon) were injected alongside mutant nonsense suppression conditions as a positive control. Data were only reported from cells that gave responses above pH 4, or cells that gave larger, more robust currents below pH 4, and other cells were considered non-responsive (NR)
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