A Conserved Salt Bridge Critical for ${\rm GABA}_{{\rm A}}$ Receptor Function and Loop C Dynamics

Chemical signaling in the brain involves rapid opening and closing of ligand gated ion channels (LGICs). LGICs are allosteric membrane proteins that transition between multiple conformational states (closed, open, and desensitized) in response to ligand binding. While structural models of cys-loop LGICs have been recently developed, our understanding of the protein movements underlying these conformational transitions is limited. Neurotransmitter binding is believed to initiate an inward capping movement of the loop C region of the ligand-binding site, which ultimately triggers channel gating. Here, we identify a critical intrasubunit salt bridge between conserved charged residues (βE153, βK196) in the ${\rm GABA}_{{\rm A}}$ receptor $({\rm GABA}_{{\rm A}}{\rm R})$ that is involved in regulating loop C position. Charge reversals (E153K, K196E) increased the EC₅₀ for GABA and for the allosteric activators pentobarbital (PB) and propofol indicating that these residues are critical for channel activation, and charge swap (E153K-K196E) significantly rescued receptor function suggesting a functional electrostatic interaction. Mutant cycle analysis of alanine substitutions indicated that E153 and K196 are energetically coupled. By monitoring disulfide bond formation between cysteines substituted at these positions (E153C-K196C), we probed the mobility of loop C in resting and ligand-bound states. Disulfide bond formation was significantly reduced in the presence of GABA or PB suggesting that agonist activation of the ${\rm GABA}_{{\rm A}}{\rm R}$ proceeds via restricting loop C mobility.