Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl⁻ into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.

Chloride ion (Cl⁻) concentration in some bacterial cells is regulated by rhodopsin proteins, generally known as halorhodopsin, or hR. These proteins use light energy to pump Cl⁻ into cells (1, 2). Light is harvested by a molecule of retinal, covalently linked to an essential lysine residue in the seventh transmembrane helix of GPCR-like (G protein–coupled receptor) proteins. Light activation causes retinal to isomerize from the all-trans to the 13-cis configuration. This change triggers subsequent conformational changes throughout the rhodopsin molecule and releases chloride into the cytoplasm. Retinal thermally relaxes to the all-trans configuration within milliseconds and is then ready for the next photocycle. Cl⁻ ions are transported from the extracellular (EC) side to the cytoplasmic (CP) side during each photocycle (3, 4).Light-driven ion-pumping rhodopsin can be used to develop artificial solar energy harvesting and optogenetics (5–8), but the molecular mechanism must be understood in detail for such applications. Despite the importance of hR, our current experimental data concerning the structure and dynamics of the protein remain very limited. A related protein, proton (H⁺)-pumping bacteriorhodopsin (bR) discovered in the early 1970s, has been extensively studied by multiple methods, including time-resolved spectroscopy, crystallography, mutagenesis, and computer simulation (9–12). In particular, recent studies using time-resolved serial femtosecond crystallography (TR-SFX) methods performed at X-ray free-electron laser (XFEL) facilities allow three-dimensional (3D) visualization of retinal isomerization and associated local conformational changes. These changes are accompanied by movement of protons from a donor aspartate group to an acceptor aspartate (13–15). However, the central component of this process, the transported H⁺, is difficult to observe by X-ray crystallography and could not be directly traced in bR TR-SFX studies. Recently, a breakthrough was reported in a study on the sodium-pumping rhodopsin KR2 (K. eikastus rhodopsin 2), in which electron density signals of Na⁺ uptake were observed at Δt = 1 ms after laser illumination (16).Cl⁻, a strong X-ray scatterer, can be directly observed from electron density maps. These maps provide first-hand information on the movement of ions as being transported within short timescales after light activation. Furthermore, hR and bR presumably share a common molecular mechanism despite transporting ions in opposite directions. A close relationship is strongly implied by the interconversion of the function of two rhodopsins. Outward H⁺-pumping bR can be converted to an inward Cl⁻ pump by changing a single residue (D85T) (17), while hR from the cyanobacterium, Mastigocladopsis repens, is reported to pump protons after a single mutation (T74D) (18). The chloride pump can therefore serve as a system analogous to the proton transporter and provide valuable information that is difficult to obtain directly from bR.In this study, we focus on chloride ion–pumping rhodopsin (ClR) from the marine flavobacterium Nonlabens marinus S1-08T (19). The conserved DTD motif (Asp85-Thr89-Asp96) of the bR family, residues 85, 89, and 96, is replaced by an NTQ motif (Asn98- Thr102-Gln109) in ClR (Fig. 1). The sequence identity of ClR and canonical bR from Halobacterium salinarum is only 27%, but the two proteins, nevertheless, have highly similar structures, including the disposition of the retinal chromophore. ClR structures at cryogenic and room temperatures clearly reveal an architecture composed of seven transmembrane helices (TM A to G) (2, 20, 21). The retinal is covalently linked to the Nζ atom of the Lys235 located on TM-G. Anomalous diffraction signals of the Br⁻ identify a stable binding site near the protonated Schiff base (PSB) and a plausible exit site on the CP side (Fig. 1A). Buried water molecules and locations of cavities inside ClR suggest a pathway for Cl⁻ uptake on the EC side, but the molecular mechanism for light-triggered Cl⁻ pumping remains obscure. Upon light activation, the Cl⁻ tightly held near the PSB must break free from its hydrogen bonding network (Fig. 1B). It then passes through a hydrophobic region to reach the CP side (Fig. 1C). Crystal structures of ClR were previously determined with crystals under continuous illumination of visible laser light. Intriguingly, these steady-state models revealed unexpected movement of the retinal, without indication of photo-isomerization (22). Steady-state measurements, which show averages of mixed states, are thus of limited use in deciphering the molecular mechanism of light-driven Cl⁻ pumping.Open in a separate windowFig. 1.Structure of ClR and a plausible pathway of Cl⁻ transport. (A) Cross-sections of ClR with the backbone structure shown in cartoon representation. Transmembrane helices are marked using letters A through G, and the C-terminal helix H in the cytoplasm is also indicated. Surfaces are clipped to show the cross-section colored in yellow and the model being sliced and then opened about the axis near the helix E. Water molecules and Cl⁻ ions are shown as red- and green-colored spheres. Blue curves indicate the path of ion entering ClR and the principal pumping direction after passing retinal. (B) Key residues near the Cl⁻ ion and retinal, together with the NTQ motif shown in stick representation. (C) Residues that form a hydrophobic region between the retinal and the cytoplasm are highlighted in ball-and-stick representation. The red arrow points to a major barrier that Cl⁻ needs to overcome. ClR backbone is shown in cartoon representation, with residues colored based on hydrophobicity (the blue to red spectrum corresponds to the hydrophobicity scale from hydrophilic to hydrophobic).     

Zoonotic infections in communities of the James Bay Cree territory: An overview of seroprevalence

The Cree communities of James Bay are at risk for contracting infectious diseases transmitted by wildlife. Data from serological testing for a range of zoonotic infections performed in the general population (six communities), or trappers and their spouses (one community), were abstracted from four population-based studies conducted in Cree territory (Quebec) between 2005 and 2009. Evidence of exposure to Trichinella species, Toxoplasma gondii, Toxocara canis, Echinococcus granulosus, Leptospira species, Coxiella burnetii and Francisella tularensis was verified in all communities, whereas antibodies against Sin Nombre virus and California serogroup viruses (Jamestown Canyon and snowshoe hare viruses) were evaluated in three and six communities, respectively. Seroprevalence varied widely among communities: snowshoe hare virus (1% to 42%), F tularensis (14% to 37%), Leptospira species (10% to 27%), Jamestown Canyon virus (9% to 24%), C burnetii (0% to 18%), T gondii (4% to 12%), T canis (0% to 10%), E granulosus (0% to 4%) and Trichinella species (0% to 1%). No subject had serological evidence of Sin Nombre virus exposure. These data suggest that large proportions of the Cree population have been exposed to at least one of the targeted zoonotic agents. The Cree population, particularly those most heavily exposed to fauna, as well as the medical staff living in these regions, should be aware of these diseases. Greater awareness would not only help to decrease exposures but would also increase the chance of appropriate diagnostic testing.     

Insulin and leptin induce Glut4 plasma membrane translocation and glucose uptake in a human neuronal cell line by a phosphatidylinositol 3-kinase- dependent mechanism

The insulin-sensitive glucose transporter Glut4 is expressed in brain areas that regulate energy homeostasis and body adiposity. In contrast with peripheral tissues, however, the impact of insulin on Glut4 plasma membrane (PM) translocation in neurons is not known. In this study, we examined the role of two anorexic hormones (leptin and insulin) on Glut4 translocation in a human neuronal cell line that express endogenous insulin and leptin receptors. We show that insulin and leptin both induce Glut4 translocation to the PM of neuronal cells and activate glucose uptake. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, totally abolished insulin- and leptin-dependent Glut4 translocation and stimulation of glucose uptake. Thus, Glut4 translocation is a phosphatidylinositol 3-kinase-dependent mechanism in neuronal cells. Next, we investigated the impact of chronic insulin and leptin treatments on Glut4 expression and translocation. Chronic exposure of neuronal cells to insulin or leptin down-regulates Glut4 proteins and mRNA levels and abolishes the acute stimulation of glucose uptake in response to acute insulin or leptin. In addition, chronic treatment with either insulin or leptin impaired Glut4 translocation. A cross-desensitization between insulin and leptin was apparent, where exposure to insulin affects leptin-dependent Glut4 translocation and vice versa. This cross-desensitization could be attributed to the increase in suppressor of cytokine signaling-3 expression, which was demonstrated in response to each hormone. These results provide evidence to suggest that Glut4 translocation to neuronal PM is regulated by both insulin and leptin signaling pathways. These pathways might contribute to an in vivo glucoregulatory reflex involving a neuronal network and to the anorectic effect of insulin and leptin.     

Low-grade systemic inflammation causes endothelial dysfunction in patients with Hashimoto's thyroiditis

OBJECTIVE: The objective of this study was to assess whether low-grade systemic inflammation might contribute to the pathogenesis of endothelial dysfunction in patients with subclinical hypothyroidism (sHT) and autoimmune thyroiditis. BACKGROUND: sHT patients are characterized by peripheral endothelial dysfunction and low-grade inflammation. METHODS: In 53 sHT and 45 healthy subjects, we studied the forearm blood flow (strain-gauge plethysmography) response to intrabrachial acetylcholine (Ach) (0.15-15 microg/min.dl) with and without local vascular COX inhibition by intrabrachial indomethacin (50 microg/min.dl) or nitric oxide synthase blockade by N-mono methyl arginine (L-NMMA) (100 microg/min.dl) or the antioxidant vitamin C (8 mg/min.dl). The protocol was repeated 2 h after systemic nonselective COX inhibition (100 mg indomethacin) or selective COX-2 blockade (200 mg celecoxib) oral administrations. RESULTS: sHT patients showed higher C-reactive protein and IL-6 values. In controls, vasodilation to Ach was blunted by L-NMMA and unchanged by vitamin C. In contrast, in sHT, the response to Ach, reduced in comparison with controls, was resistant to L-NMMA and normalized by vitamin C. In these patients, systemic but not local indomethacin normalized vasodilation to Ach and the inhibition of L-NMMA on Ach. Similar results were obtained with celecoxib. When retested after indomethacin administration, vitamin C no longer succeeded in improving vasodilation to Ach in sHT patients. Response to sodium nitroprusside was unchanged by indomethacin or celecoxib. CONCLUSIONS: In sHT patients, low-grade chronic inflammation causes endothelial dysfunction and impaired nitric oxide availability by a COX-2-dependent pathway leading to increased production of oxidative stress.     

Do inhaled carbon nanoparticles translocate directly into the circulation in humans?

RATIONALE: Increased exposure to particulate air pollution (PM(10)) is a risk factor for death and hospitalization with cardiovascular disease. It has been suggested that the nanoparticulate component of PM(10) is capable of translocating into the circulation with the potential for direct effects on the vasculature. OBJECTIVE: The study's aim was to determine the extent to which inhaled technetium-99m ((99m)Tc)-labeled carbon nanoparticles (Technegas) were able to access the systemic circulation. METHODS AND MAIN RESULTS: Ten healthy volunteers inhaled Technegas and blood samples were taken sequentially over the following 6 h. Technegas particles were 4-20 nm in diameter and aggregated to a median particle diameter of approximately 100 nm. Radioactivity was immediately detected in blood, with levels increasing over 60 min. Thin-layer chromatography of whole blood identified a species that moved with the solvent front, corresponding to unbound (99m)Tc-pertechnetate, which was excreted in urine. There was no evidence of particle-bound (99m)Tc at the origin. gamma Camera images demonstrated high levels of Technegas retention (95.6 +/- 1.7% at 6 h) in the lungs, with no accumulation of radioactivity detected over the liver or spleen. CONCLUSIONS: The majority of (99m)Tc-labeled carbon nanoparticles remain within the lung up to 6 h after inhalation. In contrast to previous published studies, thin-layer chromatography did not support the hypothesis that inhaled Technegas carbon nanoparticles pass directly from the lungs into the systemic circulation.     

Presence and distribution of ghrelin-immunopositive cells in the chicken gastrointestinal tract

The presence and distribution patterns of ghrelin, a gastric acylated peptide, were studied in the entire gastrointestinal tract of the chicken (Gallus domesticus) using the peroxidase-antiperoxidase immunohistochemical method, western blot analysis and a specific antibody against the C-terminal region of rat ghrelin. Ghrelin-immunopositive cells were observed in the mucosal layer of all segments examined. The largest numbers of ghrelin-positive cells were located at the base of lobuli of the proventriculus gland, along villi of the intestines and in crypts of the duodenum. Lower numbers of ghrelin-immunostained cells were located in crypts of jejunum and ileum and only few ghrelin-immunostained cells were detected at the base of crypts of the large intestine. Closed and open types of cells were observed in all segments. Western blot analysis confirmed the presence of ghrelin-like protein in the entire chicken gastrointestinal tract. The anatomical distribution patterns and the morphological characteristics of chicken ghrelin-positive cells suggest that they are endocrine cells. Furthermore, it is concluded that ghrelin shows a high degree of preservation during evolution from non-mammalian vertebrates to mammals.     

Misoprostol decreases oxidative stress and liver injury in bacterial lipopolysaccharide-induced endotoxemia in mice

The effect of misoprostol, a synthetic prostaglandin E1 analog, on the development of oxidative stress induced in mice with lipopolysaccharide (LPS) endotoxin was investigated. Misoprostol was administered by intraperitoneal route (i.p.) at doses of 10, 100, or 1,000 μg/kg at the time of LPS injection (200 μg/kg, i.p.). Mice were euthanized 4 h later. Lipid peroxidation (malondialdehyde; MDA), reduced glutathione (GSH), nitric oxide (nitrite/nitrate) levels as well as paraoxonase activity were measured in brain and liver. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities as well as DNA fragmentation were determined in the liver. The administration of LPS increased oxidative stress both in the brain and liver tissue. There were significantly increased MDA and nitrite and decreased GSH and PON1 activity in the brain and liver, respectively. In addition, LPS was associated with markedly elevated plasma ALT and AST level as well as increased liver DNA fragmentation. The administration of misoprostol at 100 or 1,000 μg/kg decreased brain MDA by 17.6 and 30 %, increased GSH by 29.8 and 33.3 %, and decreased nitric oxide by 21.74 and 42.5 %, respectively, compared with the lipopolysaccharide control group. Liver MDA decreased by 27 %, GSH increased by 47.7 %, and nitric oxide decreased by 37.2 % with misoprostol at 1,000 μg/kg. Paraoxonase activity increased in both the brain and liver by misoprostol administration. The increase in liver AST and ALT and DNA fragmentation after endotoxin administration was normalized by misoprostol. These results indicate that misoprostol can alleviate oxidative stress in the presence of a mild systemic inflammatory illness, indicating a new and potentially important therapeutic application for the drug.