Mutation of valine 53 at the interface between extracellular and transmembrane domains of the β principal subunit affects the GABA receptor gating.

GABA receptors (gamma-aminobutyric acid type A receptors) are pentameric ligand-gated ion channels mediating inhibition in adult mammalian brains. Their static structure has been intensely studied in the past years but the underlying molecular activatory mechanisms remain obscure. The interface between extracellular and transmembrane domains has been recognized as a key player in the receptor gating. However, the role of the valine 53 in the β1-β2 loop of the principal subunit (β) remains controversial showing differences compared to homologous residues in some cys-loop counterparts such as nAChR. To address the role of the βV53 residue in the αβγ receptor gating, we performed high resolution macroscopic and single-channel recordings. To explore underlying molecular mechanisms a variety of substituting amino acids were investigated: Glutamate and Lysine (different electric charge), Alanine (aliphatic, larger than Valine) and Histidine (same residue as in homologous αH55). We report that mutation of the βV53 residue results in alterations of nearly all gating transitions including opening/closing, preactivation and desensitization. A dramatic gating impairment was observed for glutamate substitution (βV53E) but βV53K mutation had a weak effect. The impact of histidine substitution was also small while βV53A markedly affected the receptor but to a smaller extent than βV53E. Considering available structures in desensitized and bicuculline blocked shut states we propose that strongly detrimental effect of βV53E mutation on receptor activation results from electrostatic interaction between the glutamate and βK274 on the loop M2-M3 which stabilizes the receptor in the shut state. We conclude that βV53 is strongly involved in mechanisms underlying the receptor gating.