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

GABA(A) receptors (GABA(A)Rs) 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(A)Rs was achieved but the molecular mechanisms underlying their conformational transitions remain elusive. Phenylalanine 64 (alpha 1F64) is located at the loop D of the orthosteric binding site of GABA(A)R and was found to directly interact with GABA molecule. Mutations of alpha 1F64 were demonstrated to affect not only binding but also some gating properties. Loop D is a rigid p 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 alpha 1F64 with glycine, the smallest amino acid, widely recognized as a rigidity reducer of protein structures. To this end, we assessed the impact of the alpha 1F64G mutation in the alpha 1 beta 2 gamma 2L type of GABA(A)Rs 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 (alpha 1F64L and alpha 1F64A). In particular, alpha 1F64G mutation practically abolished the desensitization process. At the same time, the alpha 1F64G 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.

Automated summary

Recent studies have shown that phenylalanine 64 (alpha 1F64) located at loop D of GABA(A) receptors plays a crucial role in the conformational transitions of the receptor, with mutations of alpha 1F64 dramatically altering gating properties.