Mutations of α1F45 residue of GABAA receptor loop G reveal its involvement in agonist binding and channel opening/closing transitions

GABA(A) receptors (GABA(A)Rs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABA(A)R static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid beta-strand belonging to an extensive beta-sheet that spans the regions involved in GABA binding and the interdomain interface which is important in receptor gating. It has been reported that loop G participates in ligand binding and gating of GABA(A)Rs, however, it remains unclear which specific gating transitions are controlled by this loop. Analysis of macroscopic responses revealed that mutation at the (alpha 1)F45 residue (loop G midpoint) resulted in slower macroscopic desensitization and accelerated deactivation. Single-channel analysis revealed that these mutations also affected open and closed times distributions and reduced open probability. Kinetic modeling demonstrated that mutations affected primarily channel opening/closing and ligand binding with a minor effect on preactivation. Thus, (alpha 1)F45 residue, in spite of its localization close to binding site, affects late gating transitions. In silico structural analysis suggested an important role of (alpha 1)F45 residue in loop G stability and rigidity as well as in general structure of the binding site. We propose that the rigid beta-sheet comprising loop G is well suited for long range communication within GABA(A)R but this mechanism becomes impaired when (alpha 1)F45 is mutated. In conclusion, we demonstrate that loop G is crucial in controlling both binding and gating of GABA(A)Rs. These data shed new light on GABA(A)R activation mechanism and may also be helpful in designing clinically relevant modulators.