γ-Aminobutyric Acid Induced Conformational Change in GABAAR Measured by Pulsed Electron Paramagnetic Resonance

Abstract

The γ-aminobutyric acid type A receptors (GABAARs) belong to the Cys-loop ligand-gated ion channel superfamily. Sharing significant homology between prokaryotic pentameric ligand-gated ion channels (pGLIC) whose structure has been determined, GABAARs consists of five homologous subunits arranged pseudo-symmetrically around a central ion pore. Each subunit consists of an extracellular domain, an intracellular domain and a transmembrane domain consisting of a loose bundle of four α helices (M1−M4), with amino acids on one face of each of the five M2 helices forming the lumen of the ion channel. As the brain's major inhibitory neurotransmitter receptor, the chloride-selective receptors are the targets for benzodiazepines, general anesthetics, anxiolytics and anticonvulsants. In a free-cysteine free background, we substituted a Cys near the top of M3 facing the lipid-protein interface. We expressed the α1β3(M286C) GABAAR in HEK293 cells, and measured the distance between spin labels by pulsed electron paramagnetic resonance using double electron electron resonance (DEER). The α1β3 (M286C) GABAARs were spin labeled when bound to the anti-flag affinity column. Purified receptors were reconstituted into lipid bilayers. The preliminary DEER data yield three distances with broad distributions at 23, 33 and 45 A. The short distance is assigned to nearest neighbor subunits and the long distance to next nearest neighbor subunits. The intermediate distance at 33 A could reflect the presence of energetically degenerate rotameric states at the labeled site. These distances establish the presence of three β-subunits in the oligomer. Upon addition of GABA, the mean long distance increases and the distance distribution narrows, suggesting that there is a decrease in the number of states upon the addition of agonist. These distances are broadly consistent with homology models based on the structure of pGLIC, Supported by GM58448 and GM35215.