α(1) Proline 277 Residues Regulate GABA(A)R Gating through M2-M3 Loop Interaction in the Interface Region

[Image: see text] Cys-loop receptors are a superfamily of transmembrane, pentameric receptors that play a crucial role in mammalian CNS signaling. Physiological activation of these receptors is typically initiated by neurotransmitter binding to the orthosteric binding site, located at the extracellular domain (ECD), which leads to the opening of the channel pore (gate) at the transmembrane domain (TMD). Whereas considerable knowledge on molecular mechanisms of Cys-loop receptor activation was gathered for the acetylcholine receptor, little is known with this respect about the GABA(A) receptor (GABA(A)R), which mediates cellular inhibition. Importantly, several static structures of GABA(A)R were recently described, paving the way to more in-depth molecular functional studies. Moreover, it has been pointed out that the TMD-ECD interface region plays a crucial role in transduction of conformational changes from the ligand binding site to the channel gate. One of the interface structures implicated in this transduction process is the M2-M3 loop with a highly conserved proline (P277) residue. To address this issue specifically for α(1)β(2)γ(2L) GABA(A)R, we choose to substitute proline α(1)P277 with amino acids with different physicochemical features such as electrostatic charge or their ability to change the loop flexibility. To address the functional impact of these mutations, we performed macroscopic and single-channel patch-clamp analyses together with modeling. Our findings revealed that mutation of α(1)P277 weakly affected agonist binding but was critical for all transitions of GABA(A)R gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α(1)P277 at the interface is strongly involved in regulating the receptor gating.