Structural studies on GABA_A receptor and glycine receptor

Abstract

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA_A receptors are inhibitory. Previously, we identified a membrane-proximal β-rich (MPBR) domain in fragment C166-L296 of GABA_A receptor α₁ subunit, forming native-like pentamers.

In the present study, another structural domain, the N-terminal (NT) domain, was shown to exist in the fragment Q28-E165. The secondary structures of both fragments were β-rich. The CD spectrum of the combined fragment Q28-L296 was additive of the spectra of the two fragments. In addition, denaturation curves of both fragments were characteristic of cooperative transitions, supporting their domain-like nature. Moreover, effects of detergent on the molecular masses of Q28-E165 and C166-L296, as monitored using laser scattering spectroscopy, indicated that intermolecular interactions were much more significant in C166-L296 than in Q28-E165.

Substitutional mutations of Cys and Trp residues in the MPBR domain suggest that these residues collectively, but not individually, contribute to the protein structure and, indirectly, to ligand binding. In particular, the residue pairs C166 and C261, C166 and W198, and C180 and W198 were found to be essential for the structure fold and consequent ligand binding. Relative positions between the residues suggested by their combined mutational effects, such as synergistic and anti-synergistic effects, are presented in a five-member ‘pin’-like model analogous to the ‘pin’ in classical Ig domains.

A fragment of glycine receptor α1 subunit homologous to the MPBR domain was over-expressed in E. coli. The recombinant fragment was found to have stable β-rich secondary structure, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies and electron microscopy suggest that the fragment forms trimers. The result is in contrast to the pentameric assembly of the equivalent GABA_A receptor fragment, indicating that sequence similarity does not necessarily imply quaternary structure similarity.

The fragments of GABA_A β₂ and γ₂ subunits homologous to NT or MPBR domains were over-expressed in E.coli. The secondary structures of these fragments were β-rich as measured using CD spectroscopy. In addition, the N-terminal fragments have more α-helix than their corresponding membrane-proximal fragments. The BZ binding capacity was compared among α₁, β₂ and γ₂ subunits.

Date of Award	2002
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology