Probing Proline Residues in the Prokaryotic Ligand-Gated Ion Channel, ELIC.
Dougherty, Dennis A
American Chemical Society
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Mosesso, R., Dougherty, D. A., & Lummis, S. (2018). Probing Proline Residues in the Prokaryotic Ligand-Gated Ion Channel, ELIC.. Biochemistry, 57 (27), 4036-4043. https://doi.org/10.1021/acs.biochem.8b00379
Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate pentameric ligand-gated ion channels (pLGICs) and has proven to be a valuable model for understanding the structure and function of this important protein family. There is nevertheless still a question about whether molecular details can be accurately extrapolated from this protein to those found in eukaryotes. Here we explore the role of proline residues (Pros) in ELIC by creating mutant receptors, expressing them in Xenopus laevis oocytes, and using whole-cell voltage-clamp electrophysiology to monitor channel activity. In contrast to eukaryotic pLGICs, proline-to-alanine (Pro-to-Ala) substitution in ELIC mostly resulted in gain of function, and even altering highly conserved Pro residues in M1 and the M2-M3 loop did not ablate function. These substitutions also mostly resulted in ablation of the modulation by Ca2+ observed in wild-type receptors. Substitution of the Pro in the "Cys loop", however, did result in nonfunctional receptors. Probing this residue with noncanonical amino acids revealed a requirement for a substituted amine at this position, as well as a general preference for Pro analogues with greater intrinsic cis biases. We propose there is likely a cis bond at the apex of the Cys loop in this protein, which is consistent with some, but not all, findings from other pLGICs. Overall, the data show that the roles of proline residues are less critical in ELIC than in other pLGICs, supporting other studies that suggest caution must be applied in using data from this prokaryotic receptor to understand molecular details of eukaryotic pLGIC receptor function.
This work was supported by National Institutes of Health Grant NS-34407 (D.A.D.) and MRC Grant MR L021676 (S.C.R.L.).
Medical Research Council (MR/L021676/1)
External DOI: https://doi.org/10.1021/acs.biochem.8b00379
This record's URL: https://www.repository.cam.ac.uk/handle/1810/282938