NarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including ...archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.
Channelrhodopsin (ChR) is a light-gated cation channel that responds to blue light. Since ChR can be readily expressed in specific neurons to precisely control their activities by light, it has ...become a powerful tool in neuroscience. Although the recently solved crystal structure of a chimeric ChR, C1C2, provided the structural basis for ChR, our understanding of the molecular mechanism of ChR still remains limited. Here we performed electrophysiological analyses and all-atom molecular dynamics (MD) simulations, to investigate the importance of the intracellular and central constrictions of the ion conducting pore observed in the crystal structure of C1C2. Our electrophysiological analysis revealed that two glutamate residues, Glu122 and Glu129, in the intracellular and central constrictions, respectively, should be deprotonated in the photocycle. The simulation results suggested that the deprotonation of Glu129 in the central constriction leads to ion leakage in the ground state, and implied that the protonation of Glu129 is important for preventing ion leakage in the ground state. Moreover, we modeled the 13-cis retinal bound; i.e., activated C1C2, and performed MD simulations to investigate the conformational changes in the early stage of the photocycle. Our simulations suggested that retinal photoisomerization induces the conformational change toward channel opening, including the movements of TM6, TM7 and TM2. These insights into the dynamics of the ground states and the early photocycle stages enhance our understanding of the channel function of ChR.
Objective: This study aimed to examine the applicability of ultrasound muscle thickness (MT) measurements for predicting whole body fat-free mass (FFM) in elderly individuals. Design and setting: ...Crosssectional study of 77 healthy elderly individuals. Methods: MTs at nine sites of the body and FFM were determined using B-mode ultrasound and dual-energy x-ray absorptiometry (DXA), respectively, in 44 women and 33 men aged 52 to 78 yrs. Stepwise multiple regression analysis produced two equations for predicting DXA-based FFM with sex (dummy: woman = 0 and man = 1) and either MTs at the anterior and posterior of thigh and lower leg (Eq1) or the product of MT and limb length (MT×LL) at thigh anterior and posterior, lower leg posterior, and upper arm anterior (Eq2) as independent variables. Results: The R2 and SEE for each of the two equations were 0.929 and 2.5 kg for Eq1 and 0.955 and 2.0 kg for Eq2. The estimated FFM from each of Eq1 (44.4 ± 8.9 kg) and Eq2 (44.4 ± 9.0 kg) did not significantly differ from that of the DXA-based FFM (44.4 ± 9.2 kg), without systematic error. However, the absolute value of the difference between the DXA-based and estimated FFM was significantly greater with Eq1 (2.0 ± 1.5 kg) than with Eq2 (1.5 ± 1.3 kg). Conclusion: The current results indicate that ultrasound MT measurement is useful to predict FFM in the elderly, and its accuracy is improved by using the product of MT and limb length as an independent variable.
Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in ...animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
Background: Channelrhodopsins, light-gated cation channels, are widely used for optogenetics.
Results: Channelrhodopsin-1/2 chimeras exhibit light-induced conformational changes with reduced Glu-129 deprotonation (Glu-90 in channelrhodopsin-2).
Conclusion: First and second channelrhodopsin-1 transmembrane helices are prerequisite for converting photostationary state of channelrhodopsin-2 into reduced desensitized state.
Significance: Channelrhodopsin-2 has some structural differences from a channelrhodopsin chimera whose x-ray crystal structure has been solved.
Ca^2＋ release from the sarcoplasmic reticulum （SR） and endoplasmic reticulum （ER） is crucial for muscle contrac- tion, cell growth, apoptosis, learning and memory. The trimeric intracellular cation ...（TRIC） channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca^2＋ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane （TM） helices with two inverted repeated regions. The electrophysiological, biochemical and biophys- ical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestra- tions are occupied with lipid molecules. This study provides the structural and functional framework for the molecu- lar mechanism of this ion channel superfamily.
Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray ...crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.
The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory ...signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500 μL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.
•Bothropic venom causes local and systemic manifestations, such as inflammation and blood incoagulability.•Plasma levels of hepatocyte growth factor were significantly increased in rats envenomated with Bothrops jararaca venom.•Similar to thrombin, metalloproteases present in B. jararaca venom process pro-hepatocyte growth factor activator.
Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream ...signalling pathways
. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with βarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.
Channelrhodopsins are microbial rhodopsins that work as light-gated ion channels. Their importance has become increasingly recognized due to their ability to control the membrane potential of ...specific cells in a light-dependent manner. This technology, termed optogenetics, has revolutionized neuroscience, and numerous channelrhodopsin variants have been isolated or engineered to expand the utility of optogenetics. Pump-like channelrhodopsins (PLCRs), one of the recently discovered channelrhodopsin subfamilies, have attracted broad attention due to their high sequence similarity to ion-pumping rhodopsins and their distinct properties, such as high light sensitivity and ion selectivity. In this review, we summarize the current understanding of the structure-function relationships of PLCRs and discuss the challenges and opportunities of channelrhodopsin research.
Biogeophysical (BGP) and biogeochemical (BGC) effects of land-use and land cover change (LULCC) are separated at the global and regional scales in new interactive CO2 simulations for the 21st ...century. Results from four earth system models (ESMs) are analyzed for the future RCP8.5 scenario from simulations with and without land-use and land cover change (LULCC), contributing to the Land-Use and Climate, IDentification of robust impacts (LUCID) project. Over the period 2006–2100, LULCC causes the atmospheric CO2 concentration to increase by 12, 22, and 66 ppm in CanESM2, MIROC-ESM, and MPI-ESM-LR, respectively. Statistically significant changes in global near-surface temperature are found in three models with a BGC-induced global mean annual warming between 0.07 and 0.23 K. BGP-induced responses are simulated by three models in areas of intense LULCC of varying sign and magnitude (between −0.47 and 0.10 K). Modifications of the land carbon pool by LULCC are disentangled in accordance with processes that can lead to increases and decreases in this carbon pool. Global land carbon losses due to LULCC are simulated by all models: 218, 57, 35 and 34 Gt C by MPI-ESM-LR, MIROC-ESM, IPSL-CM5A-LR and CanESM2, respectively. On the contrary, the CO2-fertilization effect caused by elevated atmospheric CO2 concentrations due to LULCC leads to a land carbon gain of 39 Gt C in MPI-ESM-LR and is almost negligible in the other models. A substantial part of the spread in models' responses to LULCC is attributed to the differences in implementation of LULCC (e.g., whether pastures or crops are simulated explicitly) and the simulation of specific processes. Simple idealized experiments with clear protocols for implementing LULCC in ESMs are needed to increase the understanding of model responses and the statistical significance of results, especially when analyzing the regional-scale impacts of LULCC.
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