Mutation of valine 53 at the interface between extracellular and transmembrane domains of the ß2 principal subunit affects the GABAA receptor gating.

GABAA 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 ß1-ß2 loop of the principal subunit (ß2) remains controversial showing differences compared to homologous residues in some cys-loop counterparts such as nAChR. To address the role of the ß2V53 residue in the α1ß2γ2L 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 α1H55). We report that mutation of the ß2V53 residue results in alterations of nearly all gating transitions including opening/closing, preactivation and desensitization. A dramatic gating impairment was observed for glutamate substitution (ß2V53E) but ß2V53K mutation had a weak effect. The impact of histidine substitution was also small while ß2V53A markedly affected the receptor but to a smaller extent than ß2V53E. Considering available structures in desensitized and bicuculline blocked shut states we propose that strongly detrimental effect of ß2V53E mutation on receptor activation results from electrostatic interaction between the glutamate and ß2K274 on the loop M2-M3 which stabilizes the receptor in the shut state. We conclude that ß2V53 is strongly involved in mechanisms underlying the receptor gating.

Glycine substitution of α1F64 residue at the loop D of GABAA receptor impairs gating - Implications for importance of binding site-channel gate linker rigidity.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in adult mammalian brains. In the recent years, an impressive progress in revealing the static structure of GABAARs was achieved but the molecular mechanisms underlying their conformational transitions remain elusive. Phenylalanine 64 (α1F64) is located at the loop D of the orthosteric binding site of GABAAR and was found to directly interact with GABA molecule. Mutations of α1F64 were demonstrated to affect not only binding but also some gating properties. Loop D is a rigid ß strand which seems to be particularly suitable to convey activatory signaling from the ligand binding site (LBS) to the gate at the channel pore. To test this scenario, we have investigated the substitution of α1F64 with glycine, the smallest amino acid, widely recognized as a rigidity "reducer" of protein structures. To this end, we assessed the impact of the α1F64G mutation in the α1ß2γ2L type of GABAARs on gating properties by analyzing both macroscopic responses to rapid agonist applications and single-channel currents. We found that this substitution dramatically altered all gating features of the receptor (opening/closing, preactivation and desensitization) which contrasts with markedly weaker effects of previously considered substitutions (α1F64L and α1F64A). In particular, α1F64G mutation practically abolished the desensitization process. At the same time, the α1F64G mutant maintained gating integrity manifested as single-channel activity in the form of clusters. We conclude that rigidity of the loop D plays a crucial role in conveying the activation signal from the LBS to the channel gate.