?1 Proline 277 Residues Regulate GABAAR Gating through M2-M3 Loop Interaction in the Interface Region

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 GABAA receptor (GABAAR), which mediates cellular inhibition. Importantly, several static structures of GABAAR 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 alpha 1 beta 2 gamma 2L GABAAR, we choose to substitute proline alpha 1P277 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 alpha 1P277 weakly affected agonist binding but was critical for all transitions of GABAAR gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative alpha 1P277 at the interface is strongly involved in regulating the receptor gating.

Automated summary

Cys-loop receptors are important in mammalian CNS signaling. Little is known about the GABAA receptor's activation mechanisms. Recent studies have provided structural insights into the GABAA receptor, enabling further molecular functional studies. The interface region between the transmembrane and extracellular domains plays a crucial role in transmitting conformational changes. A highly conserved proline residue within this interface is involved in regulating receptor gating.