Abstract
Prokaryotic homologues of Cys-loop receptors have proven to be useful in understanding their eukaryotic counterparts, but even the best studied of these, Gloeobacter ligand-gated ion channel (GLIC), is still not yet fully understood. GLIC is activated by protons with a pH50 between 5 and 6, implicating a histidine residue in its activation, but although a histidine residue (His11') in the pore-forming α-helix (M2) is known to be involved in gating, the His in the extracellular domain (ECD), His127, is not. Nevertheless, there is evidence from a GLIC-glycine chimera for a proton sensitive residue or region in the GLIC extracellular domain. Here we create a novel chimeric receptor with the ECD of GLIC and the transmembrane domain of ELIC (GELIC). Expression of this receptor in oocytes reveals proton activation, although the pH50 (6.7) differs from that of GLIC (5.4). Exploration of protonatable residues in the ECD reveals that the pKas of five Asp residues (31, 49, 91, 136, and 178) differ between the open and closed states of GLIC. Substitution of these residues with Ala or Asn shows somewhat similar effects for GLIC and GELIC in Asp91 mutants, but different effects for the others. Overall, the data suggest that protonation of residues in the ECD is a requirement for channel opening in GELIC but plays only a minor role in GLIC, where gating may be largely driven via protonation of the His residue in its pore.
Highlights
The discovery of pentameric ligand-gated ion channels in prokaryotes[1] has considerably enhanced our understanding of the structure and function of these proteins
To clarify if GLIC and the transmembrane domain of ELIC (GELIC) and GLIC are activated by a similar mechanism, we examined residues in the extracellular domain (ECD) that could be involved in the activation process
Proton-activated responses can be blocked by picrotoxin with an IC50 on the same order of magnitude as that of ELIC, and caffeic acid with an IC50 similar to that of GLIC, indicating that the pore and orthosteric binding site region have features that would be expected in this chimera
Summary
The discovery of pentameric ligand-gated ion channels (pLGICs) in prokaryotes[1] has considerably enhanced our understanding of the structure and function of these proteins. A His in the pore-lining M2 region (His11′) was identified some years ago as a critical activation feature,[13] and more recent data using noncanonical amino acids strongly support this residue as a, and possibly the, residue required for proton activation.[14] In addition, a chimeric protein, consisting of the ECD of ELIC and the TMD of GLIC, can be activated by protons, suggesting the GLIC activation site is in the TMD.[15] studies of a chimeric receptor with the ECD of GLIC and a glycine receptor TMD, named Lily, revealed this protein is activated by protons, suggesting a proton activation site is located in the GLIC ECD.[16,17] the Lily pH50 (pH at 50% activation) is distinct from that of GLIC, suggesting there is some difference in the details of activation of Lily and GLIC. We probe some of the features of this protein in our quest to determine if the GLIC ECD has the same role in GLIC and chimeric proteins
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