Functional asymmetry of the conserved cystine loops in αβγ GABAA receptors revealed by the response to GABA activation and drug potentiation

Abstract

Ligand-gated ion channels respond to specific neurotransmitters by transiently opening an integral membrane ion-selective pore, allowing ions to move down their electrochemical gradient. A distinguishing feature of all members of the ligand-gated ion channel superfamily is the presence of a 13-amino acid disulfide loop (Cys-loop) in the extracellular ligand-binding domain. Structural data derived from the acetylcholine receptor place this loop at the interface between the ligand-binding domain and the transmembrane pore-forming domain where it is ideally located to participate in coupling ligand binding to channel opening. We have introduced specific mutations into a conserved motif at the mid-point of the Cys-loop of the GABA A receptor subunits alpha1, beta2 and gamma2S where the sequence reads aromatic, proline, aliphatic (ArProAl motif). Receptors carrying a mutation in the Cys-loop of one of their subunits were expressed in L929 cells and responses to both GABA and drugs were assessed using the whole-cell patch clamp technique. Drug potentiation and direct activation were significantly enhanced by mutations in this Cys-loop but these effects were subunit-dependent. Currents in response to agonists were larger when mutations were carried in the alpha and beta subunits but not in the gamma subunit. In contrast, potentiation of current responses by diazepam, etomidate and pentobarbital were all enhanced when mutations were carried in the alpha and gamma subunits, but not the beta subunit. Since the disruption of interactions mediated through the ArProAl motif enhances the mutant receptor's response to both agonist and drugs we suggest that this motif in the Cys-loop of the wild-type receptor participates in interactions that create activation barriers to conformational changes during channel gating.

Protein interactions involving the γ2 large cytoplasmic loop of GABA A receptors modulate conductance

“…Standard methods were used to generate plasmid DNA. Full-length cDNAs for the wild-type human GABA A receptor subtypes ␣1 and ␤2 were subcloned individually into the pcDNA3.1 ϩ (Invitrogen, Carlsbad, CA, USA) mammalian expression vector as described previously (22). The ␥2s gene was subcloned into the multiple cloning site of the pCMS-EGFP vector (Clontech; Takara Bio Inc., Shiga, Japan).…”