Gating mechanisms of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are trimeric, proton-gated and sodium-selective members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized. Existing X-ray structures of ASIC1a describe the conformations of the open and desensitized states, but the structure of the high pH resting state and detailed mechanisms of the activation and desensitization of the channel have remained elusive. Here we present structures of the high pH resting state of homotrimeric chicken (Gallus gallus) ASIC1a, determined by X-ray crystallography and single particle cryo-electron microscopy, and present a comprehensive molecular mechanism for proton-dependent gating in ASICs. In the resting state, the position of the thumb domain is further from the three-fold molecular axis, thereby expanding the 'acidic pocket' in comparison to the open and desensitized states. Activation therefore involves 'closure' of the thumb into the acidic pocket, expansion of the lower palm domain and an iris-like opening of the channel gate. Furthermore, we demonstrate how the ß11-ß12 linkers that demarcate the upper and lower palm domains serve as a molecular 'clutch', and undergo a simple rearrangement to permit rapid desensitization.

Light-coupled cryo-plunger for time-resolved cryo-EM.

Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time-dependent conformational landscapes has been a long-standing goal of structural biology. To harness the power of single particle cryo-EM methods to enable 'time-resolved' structure determination, we have developed a light-coupled cryo-plunger that pairs flash-photolysis of caged ligands with rapid sample vitrification. The 'flash-plunger' consists of a high-power ultraviolet LED coupled with focusing optics and a motorized linear actuator, enabling the user to immobilize protein targets in vitreous ice within a programmable time window - as short as tens of milliseconds - after stimulus delivery. The flash-plunger is a simple, inexpensive and flexible tool to explore short-lived conformational states previously unobtainable by conventional sample preparation methods.

Algorithmic fairness in pandemic forecasting: lessons from COVID-19.

Racial and ethnic minorities have borne a particularly acute burden of the COVID-19 pandemic in the United States. There is a growing awareness from both researchers and public health leaders of the critical need to ensure fairness in forecast results. Without careful and deliberate bias mitigation, inequities embedded in data can be transferred to model predictions, perpetuating disparities, and exacerbating the disproportionate harms of the COVID-19 pandemic. These biases in data and forecasts can be viewed through both statistical and sociological lenses, and the challenges of both building hierarchical models with limited data availability and drawing on data that reflects structural inequities must be confronted. We present an outline of key modeling domains in which unfairness may be introduced and draw on our experience building and testing the Google-Harvard COVID-19 Public Forecasting model to illustrate these challenges and offer strategies to address them. While targeted toward pandemic forecasting, these domains of potentially biased modeling and concurrent approaches to pursuing fairness present important considerations for equitable machine-learning innovation.

The His-Gly motif of acid-sensing ion channels resides in a reentrant 'loop' implicated in gating and ion selectivity.

Acid-sensing ion channels (ASICs) are proton-gated members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout the central and peripheral nervous systems. The homotrimeric splice variant ASIC1a has been implicated in nociception, fear memory, mood disorders and ischemia. Here, we extract full-length chicken ASIC1 (cASIC1) from cell membranes using styrene maleic acid (SMA) copolymer, elucidating structures of ASIC1 channels in both high pH resting and low pH desensitized conformations by single-particle cryo-electron microscopy (cryo-EM). The structures of resting and desensitized channels reveal a reentrant loop at the amino terminus of ASIC1 that includes the highly conserved 'His-Gly' (HG) motif. The reentrant loop lines the lower ion permeation pathway and buttresses the 'Gly-Ala-Ser' (GAS) constriction, thus providing a structural explanation for the role of the His-Gly dipeptide in the structure and function of ASICs.

Divalent cation and chloride ion sites of chicken acid sensing ion channel 1a elucidated by x-ray crystallography.

Acid sensing ion channels (ASICs) are proton-gated ion channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed throughout central and peripheral nervous systems. ASICs have been implicated in multiple physiological processes and are subject to numerous forms of endogenous and exogenous regulation that include modulation by Ca2+ and Cl- ions. However, the mapping of ion binding sites as well as a structure-based understanding of the mechanisms underlying ionic modulation of ASICs have remained elusive. Here we present ion binding sites of chicken ASIC1a in resting and desensitized states at high and low pH, respectively, determined by anomalous diffraction x-ray crystallography. The acidic pocket serves as a nexus for divalent cation binding at both low and high pH, while we observe divalent cation binding within the central vestibule on the resting channel at high pH only. Moreover, neutralization of residues positioned to coordinate divalent cations via individual and combined Glu to Gln substitutions reduced, but did not extinguish, modulation of proton-dependent gating by Ca2+. Additionally, we demonstrate that anion binding at the canonical thumb domain site is state-dependent and present a previously undetected anion site at the mouth of the extracellular fenestrations on the resting channel. Our results map anion and cation sites on ASICs across multiple functional states, informing possible mechanisms of modulation and providing a blueprint for the design of therapeutics targeting ASICs.

Correction: Divalent cation and chloride ion sites of chicken acid sensing ion channel 1a elucidated by x-ray crystallography.

[This corrects the article DOI: 10.1371/journal.pone.0202134.].

Cryo-EM structure of the benzodiazepine-sensitive α1ß1γ2S tri-heteromeric GABAA receptor in complex with GABA.

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABAA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABAA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABAA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1ß1γ2SEM GABAA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABAA receptors and a framework for rational design of novel therapeutic agents.