Stanford Synchrotron Radiation Lightsource

Type A γ-aminobutyric acid receptors (GABA_ARs) control neuronal excitability¹. They are targets for the treatment of neurological diseases and disorders and also for general anesthetics. The underlying mechanisms of these drugs’ action on GABA_ARs remain to be determined.

One of the mechanisms is to potentiate function of GABA_ARs via binding to the transmembrane domain (TMD)². Ample experimental evidence suggests that the TMD of GABA_ARs harbors sites for the primary actions of general anesthetics and neurosteroids. The TMD plays an essential role in functional transitions among the resting, activated, and desensitized states of these Cl^－-conducting channels.

Alphaxalone (5α-pregnan-3α-ol-11,20 dione) is a potent neurosteroid anesthetic. The anxiolytic, anticonvulsant, analgesic, and sedative-hypnotic effects of alphaxalone have been linked to its potentiation of GABA-evoked currents and direct activation of GABA_ARs³. However, the data about the alphaxalone binding site in GABA_ARs and the underlying structural basis of alphaxalone’s action are sparse.

Figure 1. Crystal structures of the α1GABA_AR chimera in complex with alphaxalone (a) Side and (b) bottom views of the structure. The extracellular domain (ECD and transmembrane domain (TMD) are colored in yellow and orange, respectively. Alphaxalone binding to the inter-subunit sites in the TMD is indicated by the F_O-F_C omit electron density map contoured at 4 σ (green mesh). (c) A zoom-in view of alphaxalone (cyan) bound to a pocket lined by residues in the TMD from the principal (yellow) and complementary (white) subunits of the α1GABA_AR chimera. Alphaxalone is surrounded by the 2F_O-F_C electron density map contoured at 1 σ (blue mesh). Three residues in close contact with alphaxalone are highlighted.

In a recent paper published in Nature Communications (1), scientists from the University of Pittsburgh School of Medicine in collaboration with scientists from SSRL have successfully co-crystallized alphaxalone with an α1GABA_AR chimera. Their crystal structures (Figure 1) illustrate the molecular details of alphaxalone binding to the α1GABA_AR TMD and reveal neurosteroid anesthetic action starting at the bottom of the TMD.

Additionally, the study finds that alphaxalone allosterically triggers the activation and subsequent desensitization of the α1GABA_AR chimera (1). More importantly, a comparison of apo and alphaxalone-bound crystal structures reveals a set of conformational changes that suggest a plausible path for alphaxalone-induced channel activation and desensitization, starting at the bottom of the TMD (Fig. 2).

Together, the studies underscore three conclusions. First, alphaxalone binding to the inter-subunit site at the bottom of the TMD can introduce global conformational changes involved in channel potentiation, activation, and desensitization. Second, the TM1-TM2 linker, noted previously for its involvement in channel desensitization, plays a key role in mediating activation and potentiation by neurosteroids. Finally, this mechanism of activation or potentiation starting at the bottom of the TMD can be exploited in the rational search for new GABA_AR modulators with better potency and efficacy.