Tension activation of mechanosensitive two-pore domain K+ channels TRAAK, TREK-1, and TREK-2.

TRAAK, TREK-1, and TREK-2 are mechanosensitive two-pore domain K+ (K2P) channels that contribute to action potential propagation, sensory transduction, and muscle contraction. While structural and functional studies have led to models that explain their mechanosensitivity, we lack a quantitative understanding of channel activation by membrane tension. Here, we define the tension response of mechanosensitive K2Ps using patch-clamp recording and imaging. All are low-threshold mechanosensitive channels (T10%/50% 0.6-2.7 / 4.4-6.4 mN/m) with distinct response profiles. TRAAK is most sensitive, TREK-1 intermediate, and TREK-2 least sensitive. TRAAK and TREK-1 are activated broadly over a range encompassing nearly all physiologically relevant tensions. TREK-2, in contrast, activates over a narrower range like mechanosensitive channels Piezo1, MscS, and MscL. We further show that low-frequency, low-intensity focused ultrasound increases membrane tension to activate TRAAK and MscS. This work provides insight into tension gating of mechanosensitive K2Ps relevant to understanding their physiological roles and potential applications for ultrasonic neuromodulation.

Pressure and ultrasound activate mechanosensitive TRAAK K + channels through increased membrane tension.

TRAAK is a mechanosensitive two-pore domain K + (K2P) channel found in nodes of Ranvier within myelinated axons. It displays low leak activity at rest and is activated up to one hundred-fold by increased membrane tension. Structural and functional studies have led to physical models for channel gating and mechanosensitivity, but no quantitative analysis of channel activation by tension has been reported. Here, we use simultaneous patch-clamp recording and fluorescent imaging to determine the tension response characteristics of TRAAK. TRAAK shows high sensitivity and a broad response to tension spanning nearly the entire physiologically relevant tension range. This graded response profile distinguishes TRAAK from similarly low-threshold mechanosensitive channels Piezo1 and MscS, which activate in a step-like fashion over a narrow tension range. We further use patch imaging to show that ultrasonic activation of TRAAK and MscS is due to increased membrane tension. Together, these results provide mechanistic insight into TRAAK tension gating, a framework for exploring the role of mechanosensitive K + channels at nodes of Ranvier, and biophysical context for developing ultrasound as a mechanical stimulation technique for neuromodulation.

Physical basis for distinct basal and mechanically gated activity of the human K+ channel TRAAK.

TRAAK is a mechanosensitive two-pore domain K+ (K2P) channel localized to nodes of Ranvier in myelinated neurons. TRAAK deletion in mice results in mechanical and thermal allodynia, and gain-of-function mutations cause the human neurodevelopmental disorder FHEIG. TRAAK displays basal and stimulus-gated activities typical of K2Ps, but the mechanistic and structural differences between these modes are unknown. Here, we demonstrate that basal and mechanically gated openings are distinguished by their conductance, kinetics, and structure. Basal openings are low conductance, short duration, and due to a conductive channel conformation with the interior cavity exposed to the surrounding membrane. Mechanically gated openings are high conductance, long duration, and due to a channel conformation in which the interior cavity is sealed to the surrounding membrane. Our results explain how dual modes of activity are produced by a single ion channel and provide a basis for the development of state-selective pharmacology with the potential to treat disease.

Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs.

SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.

Ultrasound activates mechanosensitive TRAAK K+ channels through the lipid membrane.

Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K+ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a1,2. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma3-6. 3a has been implicated in viral release7, inhibition of autophagy8, inflammasome activation9, and cell death10,11 and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target3,12. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 Å resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.

Asymmetry of movements in CFTR's two ATP sites during pore opening serves their distinct functions.

CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, is opened by ATP binding to two cytosolic nucleotide binding domains (NBDs), but pore-domain mutations may also impair gating. ATP-bound NBDs dimerize occluding two nucleotides at interfacial binding sites; one site hydrolyzes ATP, the other is inactive. The pore opens upon tightening, and closes upon disengagement, of the catalytic site following ATP hydrolysis. Extent, timing, and role of non-catalytic-site movements are unknown. Here we exploit equilibrium gating of a hydrolysis-deficient mutant and apply Φ value analysis to compare timing of opening-associated movements at multiple locations, from the cytoplasmic ATP sites to the extracellular surface. Marked asynchrony of motion in the two ATP sites reveals their distinct roles in channel gating. The results clarify the molecular mechanisms of functional cross-talk between canonical and degenerate ATP sites in asymmetric ABC proteins, and of the gating defects caused by two common CF mutations.

Timing of CFTR pore opening and structure of its transition state.

In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation ΔF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the ΔF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in ΔF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.