Mutations of αF45 residue of GABA receptor loop G reveal its involvement in agonist binding and channel opening/closing transitions.

GABA receptors (GABARs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABAR static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid β-strand belonging to an extensive β-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 GABARs, however, it remains unclear which specific gating transitions are controlled by this loop. Analysis of macroscopic responses revealed that mutation at the αF45 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, αF45 residue, in spite of its localization close to binding site, affects late gating transitions. In silico structural analysis suggested an important role of αF45 residue in loop G stability and rigidity as well as in general structure of the binding site. We propose that the rigid β-sheet comprising loop G is well suited for long range communication within GABAR but this mechanism becomes impaired when αF45 is mutated. In conclusion, we demonstrate that loop G is crucial in controlling both binding and gating of GABARs. These data shed new light on GABAR activation mechanism and may also be helpful in designing clinically relevant modulators.