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.

RNA-seq reveals the gene expression in patterns in Populus × euramericana 'Neva' plantation under different precision water and fertilizer-intensive management.

BACKGROUND: Populus spp. is a crucial fast-growing and productive tree species extensively cultivated in the mid-latitude plains of the world. However, the impact of intensive cultivation management on gene expression in plantation remains largely unexplored. RESULTS: Precision water and fertilizer-intensive management substantially increased key enzyme activities of nitrogen transport, assimilation, and photosynthesis (1.12-2.63 times than CK) in Populus × euramericana 'Neva' plantation. Meanwhile, this management approach had a significant regulatory effect on the gene expression of poplar plantations. 1554 differential expression genes (DEGs)were identified in drip irrigation (ND) compared with conventional irrigation. Relative to ND, 2761-4116 DEGs, predominantly up-regulated, were identified under three drip fertilization combinations, among which 202 DEGs were mainly regulated by fertilization. Moreover, drip irrigation reduced the expression of cell wall synthesis-related genes to reduce unnecessary water transport. Precision drip and fertilizer-intensive management promotes the synergistic regulation of carbon and nitrogen metabolism and up-regulates the expression of major genes in nitrogen transport and assimilation processes (5 DEGs), photosynthesis (15 DEGs), and plant hormone signal transduction (11 DEGs). The incorporation of trace elements further enhanced the up-regulation of secondary metabolic process genes. In addition, the co-expression network identified nine hub genes regulated by precision water and fertilizer-intensive management, suggesting a pivotal role in regulating the growth of poplar. CONCLUSION: Precision water and fertilizer-intensive management demonstrated the ability to regulate the expression of key genes and transcription factor genes involved in carbon and nitrogen metabolism pathways, plant hormone signal transduction, and enhance the activity of key enzymes involved in related processes. This regulation facilitated nitrogen absorption and utilization, and photosynthetic abilities such as light capture, light transport, and electron transport, which faintly synergistically regulate the growth of poplar plantations. These results provide a reference for proposing highly efficient precision intensive management to optimize the expression of target genes.

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.