Functional Evaluation of Key Interactions Evident in the Structure of the Eukaryotic Cys-Loop Receptor GluCl

Abstract

The publication of the first high-resolution crystal structure of a eukaryotic Cys-loop receptor, GluClα, has provided valuable structural information on this important class of ligand-gated ion channels (LGIC). However, limited functional data exist for the GluCl receptors. Before applying the structural insights from GluCl to mammalian Cys-loop receptors such as nicotinic acetylcholine and GABA receptors, it is important to ensure that established functional features of mammalian Cys-loop receptors are present in the more distantly related GluCl receptors. Here, we seek to identify ligand-binding interactions that are generally associated with Cys-loop receptors, including the frequently observed cation−π interaction. Our studies were performed on the highly homologous GluClβ receptor, because GluClα is not activated by glutamate in Xenopus laevis oocytes. Mutagenesis of the signal peptide and pore lining helix was performed to enhance functional expression and sensitivity to applied ligand, respectively. Conventional and unnatural amino acid mutagenesis indicate a strong cation−π interaction between Y206 and the protonated amine of glutamate, as well as other important ionic and hydrogen bond interactions between the ligand and the binding site, consistent with the crystal structure.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…S2A), in line with results obtained in HEK cells (45). No responses to 500 nM IVM in CHO cells transfected with the GluClβWT subunit alone were observed (10 cells), in agreement with the same observations in HEK cells (18,22). In contrast to these differential responses, cells cotransfected with both GluClαWT and GluClβWT subunits displayed robust responses to 1.5 mM Glu (EC 50 concentration) and 500 nM IVM (Fig.…”

“…203 in ref. 22), which increased the Glu-EC 50 to a much larger extent than observed here for the heteromeric GluClαWT/βT229N or GluClαWT/βT229W receptor. This difference can be attributed to the nature of the replacing amino acids.…”

“…126 in ref. 22), the Glu-EC 50 relatively increased by 590-fold (22). Clearly, if the β-subunit (−) side were to contribute the "complementary" Glubinding components in our heteromeric GluClαWT/βS152A receptor, we would have observed a much larger rightward shift of the dose-response curve (complementary is defined in Fig.…”

“…We infer that the GluClβ(+) side in the heteromeric assemblies generated here contributes the principal Glu-binding components. Daeffler et al (22) added to the homomeric GluClβT283S receptor a β(+)T229A mutation (no. 203 in ref.…”

“…When expressed in Xenopus oocytes, homomeric GluClαRs respond to IVM but not to Glu and, in contrast, homomeric GluClβRs respond to Glu but not to IVM (13,16,17,22). A recent 3D crystal structure of a truncated homomeric GluClαR (GluClα cryst R; Protein Data Bank ID code 3RIF) shows that when IVM is bound at the five α/α intersubunit interfaces in the ion-channel pore periphery, Glu is lodged at the five α/α intersubunit interfaces in the ligand-binding domain (LigBD) (23) (Fig.…”