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

Abstract

GABA_A receptors (GABA_ARs) play a crucial role in mediating inhibition in adult mammalian brains. In the recent years, an impressive progress in revealing the static structure of GABA_ARs 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 GABA_AR 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 β 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 α1β2γ2L type of GABA_ARs 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.

Keywords: GABA(A) receptors; Gating; Loop D; Phenylalanine 64; Structure-function relationship.