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 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 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.
['Kłopotowski|Karol|K|', 'Czyżewska|Marta M|MM|', 'Mozrzymas|Jerzy W|JW|']
D000818:Animals D001665:Binding Sites D004305:Dose-Response Relationship, Drug D018755:GABA Agonists D005998:Glycine D057809:HEK293 Cells D006801:Humans D015640:Ion Channel Gating D009154:Mutation D017433:Protein Structure, Secondary D051381:Rats D011963:Receptors, GABA-A D005680:gamma-Aminobutyric Acid
Loop D Gating Phenylalanine 64 GABA(A) receptors Structure-function relationship