04 Jan 2019

What do sedatives, general anesthetics, anti-anxiety drugs, alcohol, numerous recreational drugs, and allopregnanolone, a neurosteroid being tested in Alzheimer’s trials, have in common? They all tweak the activity of GABA-A receptors. When engaged by their physiological ligand, the inhibitory neurotransmitter γ-aminobutyric acid (GABA), the receptors provide a channel for chloride ions to flood into the neuron, effectively stifling action potentials. Exactly how various drugs affect the receptors’ function has long been fuzzy, but now, a series of cryo-electron microscopy structures offers some clarity.

CryoEM structures show GABA-A receptors with GABA and modulators.

Inhibitors constrict the ion channel via allosteric mechanisms.

Benzodiazepines help GABA twist it, opening it.

Researchers led by Keith Miller of Massachusetts General Hospital in Boston and Radu Aricescu of the MRC Laboratory of Molecular Biology in Cambridge, England, reported the structures in a pair of papers in the January 2 Nature. The first shows GABA-A receptors alone; the second describes how engagement of the receptor by a handful of different compounds, including benzodiazepines and GABA itself, manages to open or constrict the ion channel.

“These papers represent an elegant set of studies that provide critical, novel insights into structure-based drug design that may facilitate development of better molecules to treat neurological diseases,” commented Celeste Karch of Washington University in St. Louis. Karch was not involved in the current work, but recently reported that GABAergic function flags in the presence of tau pathology (Dec 2018 news). Other previous studies have linked waning GABAergic function to heightened seizure activity in Alzheimer’s disease models and in people with AD (Palop et al., 2007; Nov 2009 conference news).

GABA-A receptors comprise five subunits—two α, two β, and one γ. Each is equipped with an extracellular, transmembrane, and cytosolic domain. Together, the subunits form a ring, and when the receptor is activated, this ring serves as a channel through which chloride ions pass. Just last year, two other groups used cryo-electron microscopy to elucidate structures of GABA-A receptors (Jun 2018 news; Phulera et al., 2018). Both of those studies used detergents to solubilize the transmembrane receptors for structural analysis. This protocol resulted in receptors with collapsed conformations—structures seemingly at odds with the known function of the ligand-gated ion channels.

First author Duncan Laverty and colleagues sought to visualize GABA-A receptors in a more physiological conformation. They ditched the detergent and instead used lipid nanodiscs—rings of membrane phospholipids—to stabilize the receptors. They expressed its α1β3γ2L isoform, one of the most common in the human brain, in cell lines. After isolating the receptor and reconstituting it within nanodiscs, the researchers solved its structure via single particle cryoEM.

Pentameric Channel. Side and top views of GABA-A receptor show the five subunits forming a channel through the membrane. [Courtesy of Laverty et al., Nature, 2019.]

The pentameric receptors formed a slim, cylindrical structure that passed through lipid nanodiscs. The extracellular domain of each subunit consisted of a single α-helix atop 10 β-strands, while the transmembrane region comprised four α-helices, labeled M1-4. The five M2 strands lined the inside of the channel. In contrast to the disordered transmembrane domains described in previous structures solved in the presence of detergents, the transmembrane domain of Laverty’s receptor maintained a highly ordered, quasi-symmetrical structure throughout. The intracellular domain was more of a jumble, likely due to the lack of available synaptic protein binding partners that normally dock with the receptor in the cytoplasm. Interestingly, Laverty and colleagues spotted phosphatidylinositol-4,5-bisphosphate (PIP 2 ) interacting closely with the intracellular side of the α1 subunit, an association they proposed could herd the receptors into lipid rafts within the membrane.

How would the structure change when inhibitors, GABA, or other agonists latched on? In the second paper, first author Simonas Masiulis and colleagues describe how the plant alkaloid PTX, the most widely used GABA-A inhibitor, lodged itself inside the transmembrane channel, where it made multiple contacts with surrounding M2 helices. Surprisingly, the structure indicated that rather than blocking an open pore, PTX appeared to stabilize the channel in its constricted state.

Constriction, Not Blockage. The GABA-A inhibitor PTX binds deep into the transmembrane channel, instigating its constriction at the bottom. [Courtesy of Masiulis et al., Nature, 2019.]

In contrast, the competitive antagonist bicuculline blocked the receptor by triggering closure from the outside. BCC latches onto the same β3/α1 interfaces where GABA binds. However, as opposed to the compact aromatic folds that form in response to GABA binding, BCC contorts these regions into a more open conformation. This has a trickle-down effect, completely closing the transmembrane channel when BCC is bound up above.

Benzodiazepines, on the other hand, act as GABA-A agonists. How do they help whisk ions though the channel? Masiulis and colleagues analyzed the structures of GABA-A receptors bound to GABA along with two commonly used sedatives/anxiolytics: alprazolam (Xanax), and diazepam (Valium). While GABA latched onto its binding pocket at the β3/α1 interface, the drugs wedged into the interface between α1 and γ2. GABA binding triggered changes in the β3 subunit that rotated it toward the neighboring α1 subunit, where it locked in place, and benzodiazepine binding appeared to promote this rotation.

The researchers hypothesized that the receptor works through a “lock and pull” mechanism. After GABA locked the β3 subunit to α1, the subunits pull on the other extracellular domains, rotating the entire extracellular ring counterclockwise. This twists the transmembrane domains, widening the channel.

Lock and Pull. In the proposed model, GABA (green) binding triggers a rotation of β strands, which lock in place next to neighboring subunits. Alprazolam (light blue) binding exacerbates this effect. [Courtesy of Masiulis et al., Nature, 2019.]

In an accompanying News & Views piece, Michaela Jansen of Texas Tech University Health Sciences Center in Lubbock highlighted the importance of the researchers’ nanodisc technique in preserving the natural configuration of the receptor. “Their work sheds light on the intricate network of short- and long-distance crosstalk between distinct binding sites of the receptor, and is likely to stimulate drug-discovery research." Heather Rice of KU Leuven in Belgium also commended the authors for the technical feats of the studies. “Insights gained from these structures, including binding sites of drugs and drug-induced conformational changes, provide not only a better understanding of the mechanisms of action of current GABA-A drugs but also a framework for the design of next-generation GABA-A receptor therapeutics,” she wrote.

Rice added that Alzheimer’s and other tauopathies have recently joined the list of neurological disorders characterized by GABAergic dysfunction, making modulation of the receptors an attractive therapeutic strategy.—Jessica Shugart