GABA _A receptor gating imaged on the millisecond timescale

Type-A γ-aminobutyric receptors (GABA _A Rs) are fast pentameric ligand gated ion channels (pLGICs) ^1-5 . Within a millisecond, saturating agonist concentrations trigger activity bursts consisting of high-frequency fluctuations between conductive and non-conductive states ^6,7 . These can last for tens to hundreds of milliseconds until, stochastically, receptors adopt stable, long-lived, desensitised conformations ^2,8,9 . This highly dynamic process, known as gating, controls transient passages of chloride ions across plasma membranes to enable neurotransmission and other fundamental processes in animal physiology ^10-12 . The conformational transitions inside activity bursts, inferred from electrophysiology, have remained inaccessible to structural biology investigation. Here we describe the gating motions of three GABA _A receptor variants imaged within the first 10 milliseconds of agonist application by cryogenic electron microscopy (cryo-EM) ¹³ . We illustrate how activation and desensitisation proceed through multiple asymmetric states, supported by major secondary, tertiary and quaternary structural rearrangements, and demonstrate that the same gating principles apply to both homomeric and heteromeric GABA _A Rs. Furthermore, we show that cholesterol and phospholipids stabilise newly formed inter-subunit interfaces and obstruct channel pores in short-lived desensitised states, while phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) precludes the opening of both α1β3 and α1β3γ2 GABA _A R channels. Our results provide a novel framework to interpret decades of electrophysiology observations and suggest a broadly applicable approach to investigate mechanistically the vast arrays of physiological and pharmacological modulators of GABA _A Rs ^5,14 and other fast neurotransmitter receptors ^15,16 . Moreover, the subunit interfaces and lipid-binding pockets that form and disappear during GABA _A R gating provide new opportunities to discover modulators with improved specificity and therapeutic properties.

Single-particle cryo-EM was used to explore the dynamic conformational landscape of three human GABA _A receptor variants within the first 10 milliseconds of interaction with their neurotransmitter agonists.