In Kv channels, an activation gate is thought to be located near the intracellular entrance to the ion conduction pore. Although the COOH terminus of the S6 segment has been implicated in forming the gate structure, the residues positioned at the occluding part of the gate remain undetermined. We use a mutagenic scanning approach in the Shaker Kv channel, mutating each residue in the S6 gate region (T469-Y485) to alanine, tryptophan, and aspartate to identify positions that are insensitive to mutation and to find mutants that disrupt the gate. Most mutants open in a steeply voltage-dependent manner and close effectively at negative voltages, indicating that the gate structure can both support ion flux when open and prevent it when closed. We find several mutant channels where macroscopic ionic currents are either very small or undetectable, and one mutant that displays constitutive currents at negative voltages. Collective examination of the three types of substitutions support the notion that the intracellular portion of S6 forms an activation gate and identifies V478 and F481 as candidates for occlusion of the pore in the closed state.
Ebola Zaire virus is highly pathogenic for humans, with case fatality rates approaching 90% in large outbreaks in Africa. The virus replicates in macrophages and dendritic cells (DCs), suppressing production of type I interferons (IFNs) while inducing the release of large quantities of proinflammatory cytokines. Although the viral VP35 protein has been shown to inhibit IFN responses, the mechanism by which it blocks IFN production has not been fully elucidated. We expressed VP35 from a mouse-adapted variant of Ebola Zaire virus in murine DCs by retroviral gene transfer, and tested for IFN transcription upon Newcastle Disease virus (NDV) infection and toll-like receptor signaling. We found that VP35 inhibited IFN transcription in DCs following these stimuli by disabling the activity of IRF7, a transcription factor required for IFN transcription. By yeast two-hybrid screens and coimmunoprecipitation assays, we found that VP35 interacted with IRF7, Ubc9 and PIAS1. The latter two are the host SUMO E2 enzyme and E3 ligase, respectively. VP35, while not itself a SUMO ligase, increased PIAS1-mediated SUMOylation of IRF7, and repressed Ifn transcription. In contrast, VP35 did not interfere with the activation of NF-κB, which is required for induction of many proinflammatory cytokines. Our findings indicate that Ebola Zaire virus exploits the cellular SUMOylation machinery for its advantage and help to explain how the virus overcomes host innate defenses, causing rapidly overwhelming infection to produce a syndrome resembling fulminant septic shock.
Three families of ligand-activated ion channels mediate synaptic communication between excitable cells in mammals. For pentameric channels related to nicotinic acetylcholine receptors and tetrameric channels like glutamate receptors, the pore-forming and gate regions have been studied extensively. In contrast, little is known about the structure of trimeric P2X receptor channels, a family of channels that are activated by ATP and serve crucial roles in neuronal signaling, pain transmission and inflammation. To identify the pore-forming and gate regions within P2X receptor channels, we introduced cysteine residues throughout the two transmembrane (TM) segments and studied their accessibility to thiol reactive compounds and ions. Our results show that the TM2 helix lines the central ion conduction pore, that the TM1 helix is positioned peripheral to TM2, and that the flow of ions is minimized in the closed state by a gate formed by the external region of TM2.In mammals there are seven subtypes of P2X receptor channels (termed P2X 1-7 ) that are widely expressed throughout the nervous system and many other tissues, including muscle and epithelia 1, 2 . From a structural perspective P2X receptor channels are intriguing because they are formed by three identical or related subunits 3-7 , each having a large extracellular segment of ∼ 280 amino acids that forms the ATP binding domain 8 , with two flanking transmembrane helices (TM1 and TM2) 9, 10 spanning the membrane and leaving the N and C termini on the intracellular side (Fig. 1). Our objective was to determine the relative contribution of the TMs to forming the ion conduction pore in P2X receptor channels and to localize the gate region, two fundamentally important questions that remain unresolved 1 . In the present study we set out to answer these questions by introducing cysteine (Cys) residues throughout both TMs of the P2X 2 receptor channel, an isoform that desensitizes slowly after activation by ATP 2 , and measuring the apparent rate of chemical modification with thiol reactive compounds and ions applied to the external side of the membrane. For this approach to be informative for our purposes, the thiol reactive compounds and ions should 1) not modify the channel unless a Cys is introduced, 2) modify with rates approaching those observed in aqueous solution when the channel is open, and 3) modify an introduced Cys below the gate with dramatically slower rates when the channel is closed 11-13 . These requirements were not achieved in previous Cys accessibility studies on P2X receptors 14-17 , leaving the pore-forming and gate regions unclear 1 . Although modification of residues in both TM1 and TM2 was observed, no rapid modification rates were obtained, leaving open the possibility that reagents access introduced Cys residues through non-aqueous pathways (e.g. protein or lipid) and/or react with rare conformations of the channel. Furthermore, contradictory conclusions were put forward as to the secondary structure of TM2 and the location of the ga...
The decomposition of metal nitrates in air has been systematically studied by thermogravimetry. Observed temperatures of decomposition (T d ) have been inversely correlated to the charge densities (CD) of the metal cations. Due to a back-donation of electronic cloud from the nitrate to an unfilled d-orbital of transition and noble metals, their nitrates generally exhibited lower T d s (<700 K) than those of the base metals (>850 K). The thermal stability/reducibility of metal nitrates in an hydrogen atmosphere has also been studied by temperature-programmed reduction (TPR). Observed reduction temperatures (T r ) for nitrates of the base metals and the noble metals are lower than their T d , i.e., T r < T d . The lowering of T r might be attributed to a spillover of hydrogen to a nitrate moiety through heterolytic (ionic) and homolytic (atomic) dissociation of hydrogen on the respective base and noble metals. The stoichiometry of hydrogen consumption, quantitatively measured from TPR, varied with the group of metal cations. According to the stoichiometry, the end product in the TPR reduction was NH 3 (N H 2 /N NO 3 -∼4.4) and N 2 (N H 2 /N NO 3 -∼2.4) for nitrates of the noble and base metals, respectively. The T r s for nitrates of the transition metals are often ∼20 K higher than their T d s, and the ratio N H 2 /N NO 3 -varies widely between 0.7 and 3.2. Their reduction may be triggered by thermal decomposition.
Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.
Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. Its antiviral activity was demonstrated in restricting HIV infection in vitro; however, the clinical implications remain controversial. Infection with dengue virus (DENV) is a global public health problem, and we lack an antiviral drug for DENV. Here, we evaluated the anti-DENV potential of treatment with HCQ. Immunofluorescence assays demonstrated that HCQ could inhibit DENV serotype 1-4 infection in vitro. RT-qPCR analysis of HCQtreated cells showed induced expression of interferon (IFN)-related antiviral proteins and certain inflammatory cytokines. Mechanistic study suggested that HCQ activated the innate immune signaling pathways of IFN-b, AP-1, and NFkB. Knocking down mitochondrial antiviral signaling protein (MAVS), inhibiting TANK binding kinase 1 (TBK1)/inhibitor-kB kinase e (IKKe), and blocking type I IFN receptor reduced the efficiency of HCQ against DENV-2 infection. Furthermore, HCQ significantly induced cellular production of reactive oxygen species (ROS), which was involved in the host defense system. Suppression of ROS production attenuated the innate immune activation and anti-DENV-2 effect of HCQ. In summary, HCQ triggers the host defense machinery by inducing ROS-and MAVS-mediated innate immune activation against DENV infection and may be a candidate drug for DENV infection.
P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration–response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting α-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.
Voltage-activated potassium (Kv) channels open upon membrane depolarization and proceed to spontaneously inactivate. Inactivation controls neuronal firing rates and serves as a form of short-term memory and is implicated in various human neurological disorders. Here, we use high-resolution cryo–electron microscopy and computer simulations to determine one of the molecular mechanisms underlying this physiologically crucial process. Structures of the activated Shaker Kv channel and of its W434F mutant in lipid bilayers demonstrate that C-type inactivation entails the dilation of the ion selectivity filter and the repositioning of neighboring residues known to be functionally critical. Microsecond-scale molecular dynamics trajectories confirm that these changes inhibit rapid ion permeation through the channel. This long-sought breakthrough establishes how eukaryotic K + channels self-regulate their functional state through the plasticity of their selectivity filters.
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