Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.

Structural insights into angiotensin receptor signaling modulation by balanced and biased agonists.

The peptide hormone angiotensin II regulates blood pressure mainly through the type 1 angiotensin II receptor AT1 R and its downstream signaling proteins Gq and ß-arrestin. AT1 R blockers, clinically used as antihypertensive drugs, inhibit both signaling pathways, whereas AT1 R ß-arrestin-biased agonists have shown great potential for the treatment of acute heart failure. Here, we present a cryo-electron microscopy (cryo-EM) structure of the human AT1 R in complex with a balanced agonist, Sar1 -AngII, and Gq protein at 2.9 Å resolution. This structure, together with extensive functional assays and computational modeling, reveals the molecular mechanisms for AT1 R signaling modulation and suggests that a major hydrogen bond network (MHN) inside the receptor serves as a key regulator of AT1 R signal transduction from the ligand-binding pocket to both Gq and ß-arrestin pathways. Specifically, we found that the MHN mutations N1113.35 A and N2947.45 A induce biased signaling to Gq and ß-arrestin, respectively. These insights should facilitate AT1 R structure-based drug discovery for the treatment of cardiovascular diseases.

A small-molecule activation mechanism that directly opens the KCNQ2 channel.

Pharmacological activation of voltage-gated ion channels by ligands serves as the basis for therapy and mainly involves a classic gating mechanism that augments the native voltage-dependent open probability. Through structure-based virtual screening, we identified a new scaffold compound, Ebio1, serving as a potent and subtype-selective activator for the voltage-gated potassium channel KCNQ2 and featuring a new activation mechanism. Single-channel patch-clamp, cryogenic-electron microscopy and molecular dynamic simulations, along with chemical derivatives, reveal that Ebio1 engages the KCNQ2 activation by generating an extended channel gate with a larger conductance at the saturating voltage (+50 mV). This mechanism is different from the previously observed activation mechanism of ligands on voltage-gated ion channels. Ebio1 caused S6 helices from residues S303 and F305 to perform a twist-to-open movement, which was sufficient to open the KCNQ2 gate. Overall, our findings provide mechanistic insights into the activation of KCNQ2 channel by Ebio1 and lend support for KCNQ-related drug development.

Pharmacological inhibition of Kir4.1 evokes rapid-onset antidepressant responses.

Major depressive disorder, a prevalent and severe psychiatric condition, necessitates development of new and fast-acting antidepressants. Genetic suppression of astrocytic inwardly rectifying potassium channel 4.1 (Kir4.1) in the lateral habenula ameliorates depression-like phenotypes in mice. However, Kir4.1 remains an elusive drug target for depression. Here, we discovered a series of Kir4.1 inhibitors through high-throughput screening. Lys05, the most potent one thus far, effectively suppressed native Kir4.1 channels while displaying high selectivity against established targets for rapid-onset antidepressants. Cryogenic-electron microscopy structures combined with electrophysiological characterizations revealed Lys05 directly binds in the central cavity of Kir4.1. Notably, a single dose of Lys05 reversed the Kir4.1-driven depression-like phenotype and exerted rapid-onset (as early as 1 hour) antidepressant actions in multiple canonical depression rodent models with efficacy comparable to that of (S)-ketamine. Overall, we provided a proof of concept that Kir4.1 is a promising target for rapid-onset antidepressant effects.

Structural mechanisms of TRPV2 modulation by endogenous and exogenous ligands.

The transient receptor potential vanilloid 2 (TRPV2) ion channel is a polymodal receptor widely involved in many physiological and pathological processes. Despite many TRPV2 modulators being identified, whether and how TRPV2 is regulated by endogenous lipids remains elusive. Here, we report an endogenous cholesterol molecule inside the vanilloid binding pocket (VBP) of TRPV2, with a 'head down, tail up' configuration, resolved at 3.2 Å using cryo-EM. Cholesterol binding antagonizes ligand activation of TRPV2, which is removed from VBP by methyl-ß-cyclodextrin (MßCD) as resolved at 2.9 Å. We also observed that estradiol (E2) potentiated TRPV2 activation by 2-aminoethoxydiphenyl borate (2-APB), a classic tool compound for TRP channels. Our cryo-EM structures (resolved at 2.8-3.3 Å) further suggest how E2 disturbed cholesterol binding and how 2-APB bound within the VBP with E2 or without both E2 and endogenous cholesterol, respectively. Therefore, our study has established the structural basis for ligand recognition of the inhibitory endogenous cholesterol and excitatory exogenous 2-APB in TRPV2.

Structural mechanisms for the activation of human cardiac KCNQ1 channel by electro-mechanical coupling enhancers.

The cardiac KCNQ1 potassium channel carries the important IKs current and controls the heart rhythm. Hundreds of mutations in KCNQ1 can cause life-threatening cardiac arrhythmia. Although KCNQ1 structures have been recently resolved, the structural basis for the dynamic electro-mechanical coupling, also known as the voltage sensor domain-pore domain (VSD-PD) coupling, remains largely unknown. In this study, utilizing two VSD-PD coupling enhancers, namely, the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and a small-molecule ML277, we determined 2.5-3.5 Å resolution cryo-electron microscopy structures of full-length human KCNQ1-calmodulin (CaM) complex in the apo closed, ML277-bound open, and ML277-PIP2-bound open states. ML277 binds at the "elbow" pocket above the S4-S5 linker and directly induces an upward movement of the S4-S5 linker and the opening of the activation gate without affecting the C-terminal domain (CTD) of KCNQ1. PIP2 binds at the cleft between the VSD and the PD and brings a large structural rearrangement of the CTD together with the CaM to activate the PD. These findings not only elucidate the structural basis for the dynamic VSD-PD coupling process during KCNQ1 gating but also pave the way to develop new therapeutics for anti-arrhythmia.

Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.

The organellar two-pore channel (TPC) functions as a homodimer, in which each subunit contains two homologous Shaker-like six-transmembrane (6-TM)-domain repeats. TPCs belong to the voltage-gated ion channel superfamily and are ubiquitously expressed in animals and plants. Mammalian TPC1 and TPC2 are localized at the endolysosomal membrane, and have critical roles in regulating the physiological functions of these acidic organelles. Here we present electron cryo-microscopy structures of mouse TPC1 (MmTPC1)-a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-activated Na+-selective channel-in both the apo closed state and ligand-bound open state. Combined with functional analysis, these structures provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin-slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage-sensing domain from the second 6-TM domain confers voltage dependence on MmTPC1. Endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under conditions of depolarizing membrane potential. Structural comparisons between the apo and PtdIns(3,5)P2-bound structures show the interplay between voltage and ligand in channel activation. These MmTPC1 structures reveal lipid binding and regulation in a 6-TM voltage-gated channel, which is of interest in light of the emerging recognition of the importance of phosphoinositide regulation of ion channels.

Voltage-gating and cytosolic Ca2+ activation mechanisms of Arabidopsis two-pore channel AtTPC1.

Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca2+-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca2+ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca2+ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca2+-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca2+ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.

Voltage-gated potassium channels KCNQs: Structures, mechanisms, and modulations.

KCNQ (Kv7) channels are voltage-gated, phosphatidylinositol 4,5-bisphosphate- (PIP2-) modulated potassium channels that play essential roles in regulating the activity of neurons and cardiac myocytes. Hundreds of mutations in KCNQ channels are closely related to various cardiac and neurological disorders, such as long QT syndrome, epilepsy, and deafness, which makes KCNQ channels important drug targets. During the past several years, the application of single-particle cryo-electron microscopy (cryo-EM) technique in the structure determination of KCNQ channels has greatly advanced our understanding of their molecular mechanisms. In this review, we summarize the currently available structures of KCNQ channels, analyze their special voltage gating mechanism, and discuss their activation mechanisms by both the endogenous membrane lipid and the exogenous synthetic ligands. These structural studies of KCNQ channels will guide the development of drugs targeting KCNQ channels.

Advanced LD pumped 3.3 J/1 Hz nanosecond Nd:glass preamplifier for SG-II upgrade laser facility.

We demonstrate a laser-diode-pumped multipass Nd:glass laser amplifier with a range of advanced characteristics. The amplifier exhibits high extraction efficiency, enables arbitrary shaping of spatial beam intensity, and effectively suppresses frequency modulation to amplitude modulation conversion. Our approach achieves excellent beam quality via thermal lensing and thermal depolarization compensation. When a 1.82 mJ/5 ns laser pulse was injected into the amplifier, the output energy reached up to 3.3 J with a repetition rate of 1 Hz at a central wavelength of 1053.3 nm. The near-field modulation of the amplified output beam was below 1.2, and the far-field focusing ability of the beam was 90% at 2.9 times the diffraction limit. This laser amplifier system holds potential for integration as a preamplifier within the SG-II upgrade high power laser facility.

Structures of the calcium-activated, non-selective cation channel TRPM4.

TRPM4 is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) -modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the electron cryo-microscopy structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide-binding domain and the C-terminal coiled-coil participate in the tetrameric assembly of the channel; ATP binds at the nucleotide-binding domain and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. The S1-S4 domain and the post-S6 TRP domain form the central gating apparatus that probably houses the Ca2+- and PtdIns(4,5)P2-binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and reveal the molecular architecture of the TRPM family.

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs.

Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells, its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease. Here we present the single-particle electron cryo-microscopy structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis analysis, the TRPML1 structure reveals that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) binds to the N terminus of the channel-distal from the pore-and the helix-turn-helix extension between segments S2 and S3 probably couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion-binding sites, and the conserved acidic residues form the luminal Ca2+-blocking site that confers luminal pH and Ca2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing several luminal ion passages to the pore and creating a negative electrostatic trap, with a preference for divalent cations, at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, suggesting an S4-S5 linker-mediated PtdInsP2 gating mechanism among TRPML channels.

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture.

TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.

Structure and Function of Plant and Mammalian TPC Channels.

Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. These channels are believed to be evolutionary intermediates between homotetrameric voltage-gated potassium/sodium channels and the four-domain, single subunit, voltage-gated sodium/calcium channels. Each TPC subunit contains 12 transmembrane segments that can be divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. The plant TPC channel is localized in the vacuolar membrane and is also called the SV channel for generating the slow vacuolar (SV) current observed long before its molecular identification. Three subfamilies of mammalian TPC channels have been defined - TPC1, 2, and 3 - with the first two being ubiquitously expressed in animals and TPC3 being expressed in some animals but not in humans. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and their functions are associated with various physiological processes. TPC3 is localized in the plasma membrane and its physiological function is not well defined.

Ultrasmall Pt Nanozymes Immobilized on Spherical Polyelectrolyte Brushes with Robust Peroxidase-like Activity for Highly Sensitive Detection of Cysteine.

Distinct platinum (Pt) nanozymes as peroxidase mimics have received extensive interest owing to their outstanding catalytic activity, high environmental tolerance, lower consumption, and great potential in replacing natural enzymes. However, easy agglomeration of Pt nanoparticles (Pt NPs) resulting from the high surface free energy significantly decrease their peroxidase-like activity. Herein, spherical polyelectrolyte brush (SPB)-stabilized ultrasmall Pt NPs (SPB@Pt NPs) were prepared by a novel synthetic strategy where the SPB not only performed as a nanoreactor for the synthesis of ultrasmall Pt NPs but also greatly stabilized Pt NPs against aggregation. The well-defined SPB@Pt NP nanozymes exhibited outstanding peroxidase-like activity for the catalytic oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB and were then used to establish a colorimetric sensor for rapid detection of cysteine, giving a limit of detection of 0.11 µM. Moreover, the colorimetric detection system was demonstrated with outstanding performance in sensitive and selective detection of cysteine in the presence of several interference molecules. From these results, SPB@Pt NPs have been regarded as promising peroxidase mimics for a large number of applications such as in biosensing, biomedicine, the food industry, and environmental chemistry.

Immobilization of Gold Nanoparticles in Spherical Polymer Brushes Observed by Small-Angle X-ray Scattering.

Nanosized gold nanoparticles (AuNPs) are of great interest in areas such as catalysts or imaging but are easy to aggregate due to high surface activity. To stabilize AuNPs, two approaches were employed to immobilize AuNPs in spherical polymer brushes (SPBs), namely, the in situ preparation of AuNPs within the brush layer of SPBs and external addition of preprepared citrate-capped AuNPs. The distribution and stability of AuNPs in SPBs were studied by small-angle X-ray scattering (SAXS). SAXS results demonstrated that the in situ-prepared AuNPs were mainly located on the inner layer and their amount decreased from inside to outside. In the case of external addition of preprepared AuNPs, the cationic SPB showed obvious immobilization, while almost no AuNPs were immobilized in the anionic SPB. The stable immobilization of the AuNPs in SPBs was the result of multiple interactions including complexation and electrostatic interaction. SAXS was validated to be a distinctive and powerful characterization method to provide theoretical guidance for the stable immobilization of AuNPs.

Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.

Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca(2+). Ca(2+) binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca(2+) or Ba(2+) can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Our Ba(2+)-bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels.

Circular RNA circ_0070441 regulates MPP+-triggered neurotoxic effect in SH-SY5Y cells via miR-626/IRS2 axis.

Circular RNAs (circRNAs) was suggested to play crucial regulatory roles in various human diseases, including Parkinson's disease (PD). This research aimed to investigate the function and potential mechanism of circ_0070441 in PD. MPP+ (1-methyl-4-phenylpyridinium)-treated SH-SY5Y cells was used as an in vitro cellular PD model. The expressions of circ_0070441, microRNA (miR)-626 and insulin receptor substrate 2 (IRS2) were measured by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. Cell Counting Kit-8 (CCK-8) assay, Cytotoxicity Detection Kit (Lactate Dehydrogenase), flow cytometry and Caspase-3 Assay Kit were used to detect cell viability, LDH release, cell apoptosis and caspase-3 activity, respectively. The levels of inflammation-related factors were detected by enzyme-linked immunosorbent assay (ELISA). The correlation among circ_0070441, miR-626 and IRS2 were confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. The levels of circ_0070441 and IRS2 were increased while miR-626 expression was decreased in MPP+-treated SH-SY5Y cells in dose- and time-dependent manners. Depletion of circ_0070441 alleviated MPP+-triggered neuronal damage by regulating cell apoptosis and inflammation. Circ_0070441 acted as a sponge for miR-626, and IRS2 was a target of miR-626. Besides, the neuroprotective effects of circ_0070441 knockdown or miR-626 overexpression were partly overturned by the suppression of miR-626 or IRS2 overexpression. Moreover, circ_0070441 upregulated IRS2 expression by interacting with miR-626. In summary, circ_0070441 aggravated MPP+-triggered neurotoxic effect in SH-SY5Y cells by regulating miR-626/IRS2 axis.

Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2.

In eukaryotes, alternative pre-mRNA splicing allows a single gene to encode different protein isoforms that function in many biological processes, and they are used as biomarkers or therapeutic targets for diseases. Although protein isoforms in the human genome are well annotated, we speculate that some low-abundance protein isoforms may still be under-annotated because most genes have a primary coding product and alternative protein isoforms tend to be under-expressed. A peptide coencoded by a novel exon and an annotated exon separated by an intron is known as a novel junction peptide. In the absence of known transcripts and homologous proteins, traditional whole-genome six-frame translation-based proteogenomics cannot identify novel junction peptides, and it cannot capture novel alternative splice sites. In this article, we first propose a strategy and tool for identifying novel junction peptides, called CJunction, which we then integrate into a proteogenomics process specifically designed for novel protein isoform discovery and apply to the analysis of a deep-coverage HeLa mass spectrometry data set with identifier PXD004452 in ProteomeXchange. We succeeded in identifying and validating three novel protein isoforms of two functionally important genes, NHSL1 (causative gene of Nance-Horan syndrome) and EEF1B2 (translation elongation factor), which validate our hypothesis. These novel protein isoforms have significant sequence differences from the annotated gene-coding products introduced by the novel N-terminal, suggesting that they may play importantly different functions.