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Proteins are biological machines composed of a single or multiple threads of amino acids which fold into three-dimensional shapes. These shapes and surfaces directly participate in many of the molecular interactions essential to life. Some proteins exist in lipid membranes and thus have been termed membrane proteins. Membranes serve as sites of import, export, and signaling and allow for compartmentalization; all critical functions for living organisms. After decades of research throughout the 20th century, many families of membrane proteins were revealed to form transport proteins that facilitate movement of substrates across membranes. The general group of transport proteins can be further divided into two groups: active and passive transporters. Passive transport proteins have been called channels, given their observed activity of opening to allow substrate diffusion down an electrochemical gradient. Channels are diverse both in their architecture and substrates, which include ions, small molecules, and other proteins. Despite the broad importance of channels in a vast array of biology many aspects of how these machines operate at a molecular level are yet to be revealed. Determining the structures of channels, which in many cases was not technically feasible until recently, has provided key insights into the form and function of these intricate machines. Presented here are studies of two different channels: the Translocase of the Outer Mitochondrial Membrane (TOM complex) and the light-gated ion channel, ChRmine. The first study in this thesis examines the long-awaited architecture and transport mechanisms of the major protein gate of the outer mitochondrial membrane. The high-resolution structure of the TOM complex revealed its overall organization-- including all its core subunits, multiple assemblies of the complex, and a glimpse at the pore which provides insights into mechanisms of protein transport. The subsequent study examines the first ever structures of a light-gated ion channel in a native-like membrane environment, using lipid nanodiscs and cryogenic electron-microscopy(cryo-EM). Determining cryo-EM structures of ChRmine was a technical achievement and revealed its surprising trimeric assembly, ion conduction path, and retinal binding pocket. Through analysis of the structure, we were able to tune the kinetics of ChRmine using a rational engineering approach. Lastly, the latest work on ChRmine paves the path for the determination of structures previously too dynamic to capture. These elusive structures are attractive targets because they could lay the foundation for building the next generation of tools for studying the brain.

The endoplasmic reticulum (ER) is a major site for protein biosynthesis, required for production of about a third of all proteins in eukaryotic cells. The heterotrimeric Sec61 complex is a protein-conducting channel which mediates the transport or membrane integration of a majority of the ER-targeted proteins, such as secretory proteins and membrane proteins. Using a sophisticated gating mechanism, Sec61 transports soluble amino acid segments through its vertical water filled pore, and integrates hydrophobic transmembrane helices laterally into the membrane. The Sec61 channel translocates client polypeptides either co- or post-translationally. The posttranslational mode requires the formation of a larger complex with the membrane proteins Sec62 and Sec63. Substrate translocation further requires the essential ER resident Hsp70, BiP. The post translational complex in fungal species is additionally associated with the non-essential proteins Sec71 and Sec72. The structure and mechanism by which these proteins activate Sec61 for transport of client proteins has been poorly understood.Using cryo-electron microscopy (cryo-EM), we determined the structures of several variants of the Sec61–Sec62–Sec63–Sec71–Sec72 complex (shortly, the Sec complex) from Saccharomyces cerevisiae and Thermomyces lanuginosus and showed that Sec62 and Sec63 induce opening of the Sec61 channel. The structures reveal that Sec63 is positioned in the back of Sec61 and interacts with Sec61 and Sec71-Sec72, while Sec62 is flexibly associated with the complex in front of the lateral gate. The J-domain of Sec63 is optimally positioned under the channel pore to bind BiP and enable efficient polypeptide translocation. Sec63 associates with Sec61 tightly through interactions in cytosolic, transmembrane, and ER-luminal domains. These extensive interactions pry open Sec61’s lateral gate. Association of Sec62 further opens the luminal end of the lateral gate causing dislocation of the small helix blocking the pore called the plug domain. A simultaneous disruption of the cytosolic and luminal interactions of Sec63 with Sec61 completely closes the channel and abolishes its binding with Sec62. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our studies show how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.

Protein tunnels are essential in transporting small molecules into the active sites of enzymes. Tunnels' geometrical and physico-chemical properties influence the transport process. The tunnels are attractive hot spots for protein engineering and drug development. However, studying the ligand binding and unbinding using experimental techniques is challenging, while in silico methods come with their limitations, especially in the case of resource-demanding virtual screening pipelines. Caver Web 1.2 is a new version of the web server combining the capabilities for the detection of protein tunnels with the calculation of the ligand trajectories. The new version of the Caver Web server was expanded with the ability to fetch novel ligands from the Integrated Database of Small Molecules and with the fully automated virtual screening pipeline allowing for the fast evaluation of the predefined set of over 4,300 currently approved drugs. The virtual screening pipeline is accompanied by a comprehensive user interface, making it a viable service for the broader spectrum of companies and the academic user community. The web server is freely available for academic use at https://loschmidt.chemi.muni.cz/caverweb.

Due to the unpredictable presence of Non-Cognitive Users (NCUs) in the time and frequency domains, the number of available channels (i.e., channels where no NCUs exist) and corresponding channel indices per Cognitive User (CU) may differ. In this paper, we propose a heuristic channel allocation method referred to as Enhanced Multi-Round Resource Allocation (EMRRA), which employs the asymmetry of available channels in existing MRRA to randomly allocate a CU to a channel in each round. EMRRA is designed to enhance the overall spectral efficiency and fairness of channel allocation. To do this, the available channel with the lowest redundancy is primarily selected upon allocating a channel to a CU. In addition, when there are multiple CUs with the same allocation priority, the CU with the smallest number of available channels is chosen. We execute extensive simulations in order to investigate the effect of the asymmetry of available channels on CUs and compare the performance of EMRRA to that of MRRA. As a result, in addition to the asymmetry of available channels, it is confirmed that most of the channels are simultaneously available to multiple CUs. Furthermore, EMRRA outperforms MRRA in terms of the channel allocation rate, fairness, and drop rate and has a slightly higher collision rate. In particular, EMRRA can remarkably reduce the drop rate compared to MRRA.

Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference-based crop protection solutions. Asymmetrical flow field -flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the perfor-mance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75-6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allow-ing separation to be combined with data collection on yield as well as molecular weight ( MW ) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules.(c) 2022 The Author(s). Published by Elsevier B.V.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Background: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants. Hypothesis: We hypothesized that in SARS-CoV-2, two highly conserved Orf3a and E channels directly related to the virus replication were a target for the detection and inhibition of the viral replication, independent of the variant, using FDA-approved ion channel modulators. Methods: A combination of a fluorescence potassium ion assay with channel modulators was developed to detect SARS-CoV-2 Orf3a/E channel activity. Two FDA-approved drugs, amantadine (an antiviral) and amitriptyline (an antidepressant), which are ion channel blockers, were tested as to whether they inhibited Orf3a/E channel activity in isolated virus variants and in nasal swab samples from COVID-19 patients. The variants were confirmed by PCR sequencing. Results: In isolated SARS-CoV-2 Alpha, Beta, and Delta variants, the channel activity of Orf3a/E was detected and inhibited by emodin and gliclazide (IC50 = 0.42 mM). In the Delta swab samples, amitriptyline and amantadine inhibited the channel activity of viral proteins, with IC50 values of 0.73 mM and 1.11 mM, respectively. In the Omicron swab samples, amitriptyline inhibited the channel activity, with an IC50 of 0.76 mM. Conclusions: We developed an efficient method to screen FDA-approved ion channel modulators that could be repurposed to detect and inhibit SARS-CoV-2 viral replication, independent of variants.

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.

The periodic flexural-gravity waves propagating along a frozen channel are investigated. The channel has a rectangular cross section. The fluid in the channel is inviscid, incompressible and covered with ice. The ice is modeled by a thin elastic plate whose thickness varies linearly. Two cases have been considered: the ice thickness varies symmetrically across the channel, being the smallest at the center of the channel and the largest at the channel walls; the ice thickness varies from the smallest value at the one wall to the largest value at another wall. The periodic 2D problem is reduced to the problem of the wave profiles across the channel. The solution of the last problem is obtained by the normal mode method of an elastic beam with linear thickness. The behavior of flexural-gravity waves depending on the inclination parameter of the ice thickness has been studied and the results have been compared with those for a constant-thickness plate. Dispersion relations, profiles of flexural-gravity waves across the channel and distributions of strain in the ice cover have been determined. In the asymmetric case, it is shown that for long waves, the most probable plate failure corresponds to transverse strains at the thin edge of the plate, which can lead to detachment of the ice from the corresponding bank. For short waves, the longitudinal stresses within the plate, localized closer to the thick edge, become maximum. This can lead to cracking of the plate in transverse direction. In the symmetric case, the maximum strains are achieved inside the plate—close to the center, but not necessarily in the midpoint.

The complexity of neurodegenerative diseases, among which Alzheimer's disease plays a pivotal role, poses one of the tough therapeutic challenges of present time. In this perspective, a multitarget approach appears as a promising strategy to simultaneously interfere with different defective pathways. In this paper, a structural simplification plan was performed on our previously reported multipotent polycyclic compounds, in order to obtain a simpler pharmacophoric central core with improved pharmacokinetic properties, while maintaining the modulating activity on neuronal calcium channels and glycogen synthase kinase 3-beta (GSK-3β), as validated targets to combat Alzheimer's disease. The molecular pruning approach applied here led to tetrahydroisoindole-dione (1), tetrahydromethanoisoindole-dione (2) and tetrahydroepoxyisoindole-dione (3) structures, easily affordable by Diels-Alder cycloaddition. Preliminary data indicated structure 3 as the most appropriate, thus a SAR study was performed by introducing different substituents, selected on the basis of the commercial availability of the furan derivatives required for the synthetic procedure. The results indicated compound 10 as a promising, structurally atypical, safe and BBB-penetrating Cav modulator, inhibiting both L- and N-calcium channels, likely responsible for the Ca2+ overload observed in Alzheimer's disease. In a multitarget perspective, compound 11 appeared as an effective prototype, endowed with improved Cav inhibitory activity, with respect to the reference drug nifedipine, and encouraging modulating activity on GSK-3β.

This research studies the problem of a joint capacity/capacity reliability analysis of the multiuser multi-input multioutput (MIMO) system functioning in the presence of generalized multipath fading. The study presents the derived results of the closed-form analytical statistical description of the ergodic sum-rate capacity, the capacity reliability and the capacity’s higher-order statistics in the case of complex Nakagami-m distributed channel transmission coefficients. A numerical verification of the derived expressions was performed, and it demonstrated excellent correspondence with the simulation. The system performance was evaluated with the help of a numerical analysis of the joint first- and second-order statistics description, depending on the channel and system parameters. The results demonstrated several peculiarities, e.g., the existence of a specific extremum of the capacity reliability for small-sized MIMO systems, its opposite behavior (in terms of the varying number of antenna elements) for heavy and light fading, and the existing asymptotic regions of the system and the channel parameters.

The use of unmanned aerial vehicles (UAV) to provide services such as the Internet, goods delivery, and air taxis has become a reality in recent years. The use of these aircraft requires a secure communication between the control station and the UAV, which demands the characterization of the communication channel. This paper aims to present a measurement setup using an unmanned aircraft to acquire data for the characterization of the radio frequency channel in a propagation environment with particular vegetation (Caatinga) and a lake. This paper presents the following contributions: identification of the communication channel model that best describes the characteristics of communication; characterization of the effects of large-scale fading, such as path loss and log-normal shadowing; characterization of small-scale fading (multipath and Doppler); and estimation of the aircraft speed from the identified Doppler frequency.

The observed precipitation was suggestive of abundant precipitation in upstream Qilian mountains and low precipitation in the downstream oasis and desert in an endorheic basin. However, precipitation in mountains generated from the recycled moisture over oasis and desert areas has rarely been studied. The climatological patterns of water vapor from 1980 to 2017 in the Qilian Mountain Region (QMR) and Hexi Corridor Region (HCR) were investigated by the European Centre for Medium-Range Weather Forecasts Interim reanalysis dataset and the Modern-Era Retrospective Analysis for Research and Application, Version 2 reanalysis dataset. The results suggest that the precipitable water content decreases from the adjacent to the mountain areas. There are two channels that transport water vapor from the HCR to the QMR in the low troposphere (surface—600 hPa), suggesting that parts of recycled moisture generated from evapotranspiration over the oasis and desert of the HCR is transported to the QMR, contributing to the abundant precipitation in the QMR. This indicates that the transport mechanism is probably because of the “cold and wet island effect” of the cryosphere in QMR. This is likely one of the essential mechanisms of the water cycle in endorheic river basins, which has rarely been reported.

Asymmetrical flow field-flow (AF4) fractionation aims in separation of sample components to yield elution of homogenous fractions identified as well-defined peaks in the chromatograms. Separation that occurs in matrixfree open channel potentiates high recovery that can be close to 100%. However, sample properties and separation conditions may induce carryover of sample components during AF4 analysis and in sample sequences. This compromises the quality of the data collected from the online detectors and the downstream offline analytics of the collected fractions. In this study, we followed sample carryover in AF4 using model viruses and analyzed various cleaning solutions and rinse methods to reduce carryover. We introduce an SDS-NaOH -based rinsing and decontamination protocol for the AF4 instrument enabling high-quality data collection.

Whether the mitochondrial permeability transition pore (PTP), also called mitochondrial megachannel (MMC), originates from the F-ATP synthase is a matter of controversy. This hypothesis is supported both by site-directed mutagenesis of specific residues of F-ATP synthase affecting regulation of the PTP/MMC and by deletion of specific subunits causing dramatic changes in channel conductance. In contrast, human cells lacking an assembled F-ATP synthase apparently display persistence of the PTP. We discuss recent data that shed new light on this controversy, supporting the conclusion that the PTP/MMC originates from a Ca2+ -dependent conformational change in F-ATP synthase allowing its reversible transformation into a high-conductance channel.

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, &gt, 20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Clavus davidgilmouri Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.

Donors of nitroxyl (HNO), the one electron-reduction product of nitric oxide (NO.), positively modulate cardiac contractility/relaxation while limiting ischemia-reperfusion (I/R) injury. The mechanisms underpinning HNO anti-ischemic effects remain poorly understood. Using isolated perfused rat hearts subjected to 30 min global ischemia/1 or 2 h reperfusion, here we tested whether, in analogy to NO., HNO protection requires PKCε translocation to mitochondria and KATP channels activation. To this end, we compared the benefits afforded by ischemic preconditioning (IPC; 3 cycles of I/R) with those eventually granted by the NO. donor, diethylamine/NO, DEA/NO, and two chemically unrelated HNO donors: Angeli’s salt (AS, a prototypic donor) and isopropylamine/NO (IPA/NO, a new HNO releaser). All donors were given for 19 min before I/R injury. In control I/R hearts (1 h reperfusion), infarct size (IS) measured via tetrazolium salt staining was 66 ± 5.5% of the area at risk. Both AS and IPA/NO were as effective as IPC in reducing IS [30.7 ± 2.2 (AS), 31 ± 2.9 (IPA/NO), and 31 ± 0.8 (IPC), respectively)], whereas DEA/NO was significantly less so (36.2 ± 2.6%, p < 0.001 vs. AS, IPA/NO, or IPC). IPA/NO protection was still present after 120 min of reperfusion, and the co-infusion with the PKCε inhibitor (PKCV1-2500 nM) prevented it (IS = 30 ± 0.5 vs. 61 ± 1.8% with IPA/NO alone, p < 0.01). Irrespective of the donor, HNO anti-ischemic effects were insensitive to the KATP channel inhibitor, 5-OH decanoate (5HD, 100 μM), that, in contrast, abrogated DEA/NO protection. Finally, both HNO donors markedly enhanced the mitochondrial permeability transition pore (mPTP) ROS threshold over control levels (≅35–40%), an action again insensitive to 5HD. Our study shows that HNO donors inhibit mPTP opening, thus limiting myocyte loss at reperfusion, a beneficial effect that requires PKCε translocation to the mitochondria but not mitochondrial K+ channels activation.

Chloroplasts are endosymbiotic organelles and play crucial roles in energy supply and metabolism of eukaryotic photosynthetic organisms (algae and land plants). They harbor channels and transporters in the envelope and thylakoid membranes, mediating the exchange of ions and metabolites with the cytosol and the chloroplast stroma and between the different chloroplast subcompartments. In secondarily evolved algae, three or four envelope membranes surround the chloroplast, making more complex the exchange of ions and metabolites. Despite the importance of transport proteins for the optimal functioning of the chloroplast in algae, and that many land plant homologues have been predicted, experimental evidence and molecular characterization are missing in most cases. Here, we provide an overview of the current knowledge about ion and metabolite transport in the chloroplast from algae. The main aspects reviewed are localization and activity of the transport proteins from algae and/or of homologues from other organisms including land plants. Most chloroplast transporters were identified in the green alga Chlamydomonas reinhardtii, reside in the envelope and participate in carbon acquisition and metabolism. Only a few identified algal transporters are located in the thylakoid membrane and play role in ion transport. The presence of genes for putative transporters in green algae, red algae, diatoms, glaucophytes and cryptophytes is discussed, and roles in the chloroplast are suggested. A deep knowledge in this field is required because algae represent a potential source of biomass and valuable metabolites for industry, medicine and agriculture. Electronic supplementary material The online version of this article (10.1007/s00018-018-2793-0) contains supplementary material, which is available to authorized users.

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1-3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [P-32]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration.

The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs. ispartof: PLOS ONE vol:13 issue:12 ispartof: location:United States status: published

A new family of tandem facial glucosides/maltosides (TFGs/TFMs) for membrane protein manipulation was prepared. The best detergent varied depending on the hydrophobic thickness of the target protein, but ether‐based TFMs (TFM‐C0E, TFM‐C3E, and TFM‐C5E) were notable for their ability to confer higher membrane protein stability than the previously developed amide‐based TFA‐1 (P. S. Chae, K. Gotfryd, J. Pacyna, L. J. W. Miercke, S. G. F. Rasmussen, R. A. Robbins, R. R. Rana, C. J. Loland, B. Kobilka, R. Stroud, B. Byrne, U. Gether, S. H. Gellman, J. Am. Chem. Soc. 2010, 132, 16750–16752). Thus, this study not only introduces novel agents with the potential to be used in membrane protein research but also highlights the importance of both the hydrophobic length and linker functionality of the detergent in stabilizing membrane proteins.

Recent advances in cryo-electron microscopy (cryo-EM) have made it possible to solve structures of biological macromolecules at near atomic resolution. Development of more stable microscopes, improved direct electron detectors and faster software for image processing has enabled structural solution of not only large macromolecular (megadalton range) complexes but also small (~60 kDa) proteins. As a result of the widespread use of the technique, we have also witnessed new developments of techniques for cryo-EM grid preparation of membrane protein samples. This includes new types of solubilization strategies that better stabilize these protein complexes and the development of new grid supports with proven efficacy in reducing the motion of the molecules during electron beam exposure. Here, we discuss the practicalities and recent challenges of membrane protein sample preparation and vitrification, as well as grid support and foil treatment in the context of the structure determination of protein complexes by single particle cryo-EM.

The permeability transition pore (PTP) is a Ca2+‐dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)‐ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP‐dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch‐clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F‐ATP synthase.

The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

Bacterial mechanosensitive channel of small conductance (MscS) is a protein, whose activity is modulated by membrane tension, voltage and cytoplasmic crowding. MscS is a homoheptamer and each monomer consists of three transmembrane helices (TM1-3). Hydrophobic pore of the channel is made of TM3s surrounded by peripheral TM1/2s. MscS gating is a complex process, which involves opening and inactivation in response to the increase of membrane tension. A number of MscS mutants were isolated. Among them mutants affecting gating have been found including gain-of-function (GOF) and loss-of-function (LOF) that open at lower or at higher thresholds, respectively. Previously, using an in vivo screen we isolated multiple MscS mutants that leak potassium and some of them were GOF or LOF. Here we show that for a subset of these mutants K+ leak is negatively (NTD) or positively (PTD) temperature dependent. We show that temperature reliance of these mutants does not depend on how MS gating is affected by a particular mutation. Instead, we argue that NTD or PTD leak is due to the opposite allosteric coupling of the structures that determine the temperature dependence to the channel gate. In PTD mutants an increased hydration of the pore vestibule is directly coupled to the increase in the channel conductance. In NTD mutants, at higher temperatures an increased hydration of peripheral structures leads to complete separation of TM3 and a pore collapse.

The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, we identified stromal interaction molecule 1 precursor (STIM1) as an auxiliary protein of this epithelial Ca2+channel using confocal microscopy analysis and GST pull-down assay. The STIM1 protein associates specifically with the C-terminal tail of TRPV4 to form a complex. In previous reports, we demonstrated that the serine824 residue of TRPV4 is one of the target phosphorylation sites of serum/glucocorticoid regulated kinase 1 (SGK1). In this report we further identified the role of serine 824 phosphorylation. The TRPV4 mutant S824D (not S824A) exhibited a diminished capacity to bind STIM1. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STIM1 is part of the TRPV4 protein complex. Our observations clearly suggest that the formation of a complex between TRPV4 and STIM1 and its plasma membrane localization are regulated through phosphorylation of serine824 of TRPV4, and that the STIM1-TRPV4 complex plays crucial roles in routing TRPV4 to the plasma membrane from the endoplasmic reticulum and in maintaining its function.

Alzheimer's disease severely compromises cognitive function. One of the mechanisms to explain the pathology of Alzheimer’s disease has been the hypotheses of amyloid-pore/channel formation by complex Aβ-aggregates. Clinical studies suggested the moderate alcohol consumption can reduces probability developing neurodegenerative pathologies. A recent report explored the ability of ethanol to disrupt the generation of complex Aβ in vitro and reduce the toxicity in two cell lines. Molecular dynamics simulations were applied to understand how ethanol blocks the aggregation of amyloid. On the other hand, the in silico modeling showed ethanol effect over the dynamics assembling for complex Aβ-aggregates mediated by break the hydrosaline bridges between Asp 23 and Lys 28, was are key element for amyloid dimerization. The amyloid pore/channel hypothesis has been explored only in neuronal models, however recently experiments suggested the frog oocytes such an excellent model to explore the mechanism of the amyloid pore/channel hypothesis. So, the used of frog oocytes to explored the mechanism of amyloid aggregates is new, mainly for amyloid/pore hypothesis. Therefore, this experimental model is a powerful tool to explore the mechanism implicates in the Alzheimer’s disease pathology and also suggests a model to prevent the Alzheimer’s disease pathology. [BMB Reports 2015; 48(1): 13-18]

Anion transporters play a vital role in cellular processes and their dysregulation leads to a range of diseases such as cystic fibrosis, Bartters syndrome and epilepsy. Synthetic chloride transporters are known to induce apoptosis in cancer cell lines. Herein, we report triamide macrocycles that are easily synthesized and externally functionalized by pendant membrane-permeable groups. Among a variety of chains appended onto the macrocycle scaffold, cholesterol is found to be the best with an EC50 value of 0.44 mu M. The macrocycle is highly anion-selective and transports ions via an OH-/X- antiport mechanism. The macrocycle is an interesting scaffold for ion-transport as it is able to discriminate between various anions and shows a preference for SCN- and Cl-. Such anion-selective transporters are highly attractive model systems to study ion-transport mechanisms and could potentially be of high therapeutic value.

International audience; The transport of proteins at the cell surface of Bacteriodetes depends on a secretory apparatus known as Type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins such as adhesins required for gliding motility in Flavobacteria, S-layer components in Tannerella forsythia and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. While a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed, and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

F‐ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca2+‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca2+‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca2+‐ATP hydrolysis, confer resistance to Ca2+‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.

Protein conformations play crucial roles in most, if not all, biological processes. Here we show that the current carried through a nanopore by ions allows monitoring conformational changes of native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer (smFRET) and ensemble measurements revealed that proteins trapped inside the nanopore have bulk-like properties. Two ligand-free and two ligand-bound conformations of SBD proteins were inferred and their kinetic constants were determined. Remarkably, internalized proteins aligned with the applied voltage bias, and their orientation could be controlled by the addition of a single charge to the protein surface. Nanopores can thus be used to immobilize proteins on a surface with a specific orientation, and will be employed as nanoreactors for single-molecule studies of native proteins. Moreover, nanopores with internal protein adaptors might find further practical applications in multianalyte sensing devices.

Aquaporins (AQPs) are membrane water/glycerol channels that are involved in many physiological functions. Aquaporin-based modulators are predicted to have potential utility in the treatment of several diseases, as well as chemical tools to assess AQPs function in biological systems. We recently reported gold(III) compounds as human AQP3 inhibitors, with Auphen as the most potent of the series. In this work, we assessed the modulation of aquaporin-7 (AQP7) expressed in an adipocyte cell model and show that Auphen significantly inhibits mouse and human AQP7. By homology modeling and molecular docking it was possible to identify the thioether groups of methionine residues, in particular Met47, as likely candidates for binding to the gold(III) complex. Our data point to Auphen as a useful chemical tool to detect AQP7 function. It might constitute a basis to develop inhibitors with improved affinity towards different aquaglyceroporin isoforms.

The large-pore channels formed by the pannexin family of proteins have been implicated in many physiological and pathophysiological functions, mainly through their ATP release function. However, a tight regulation of channel opening is necessary to modulate their function in vivo. Post-translational modifications have been postulated as some of the regulating mechanisms for Panx1, while Panx2 and Panx3 have not been as well characterized. Positive regulators include caspase cleavage to open Panx1 channels in apoptotic cells, and activation by Src family kinases via ionotropic receptors in neurons and macrophages. S-nitrosylation of cysteines has been shown to both inhibit and activate the Panx1 channel in different cell types. All three pannexins are N-glycosylated but to different levels of modification. Their diverse glycosylation appears to regulate cellular localization, intermixing, and may restrict their ability to function as inter-cellular channels. It is clear that our understanding of pannexin post-translational modification and their role in channel function regulation is still in its infancy even a decade after their discovery.

Water is clearly important for the functioning of Photosystem II (PSII). Apart from being the very substrate that needs to be transported in this water oxidation enzyme, water is also vital for the transport of protons to and from the catalytic center as well as other important co-factors and key residues in the enzyme. The latest crystal structural data of PSII have enabled detailed analyses of the location and possible function of water molecules in the enzyme. Significant progress has also been made recently in the investigation of channels and pathways through the protein complex. Through these studies, the mechanistic significance of water for PSII is becoming increasingly clear. An overview and discussion of key aspects of the current research on water in PSII is presented here. The role of water in three other systems (aquaporin, bacteriorhodopsin and cytochrome P450) is also outlined to illustrate further points concerning the central significance that water can have, and potential applications of these ideas for continued research on PSII. It is advocated that water be seen as an integral part of the protein and far from a mere solvent.

Sea anemones&rsquo, venom is rich in peptides acting on different biological targets, mainly on cytoplasmic membranes and ion channels. These animals are also a source of pancreatic &alpha, amylase inhibitors, which have the ability to control the glucose level in the blood and can be used for the treatment of prediabetes and type 2 diabetes mellitus. Recently we have isolated and characterized magnificamide (44 aa, 4770 Da), the major &alpha, amylase inhibitor of the sea anemone Heteractis magnifica mucus, which shares 84% sequence identity with helianthamide from Stichodactyla helianthus. Herein, we report some features in the action of a recombinant analog of magnificamide. The recombinant peptide inhibits porcine pancreatic and human saliva &alpha, amylases with Ki&rsquo, s equal to 0.17 &plusmn, 0.06 nM and 7.7 &plusmn, 1.5 nM, respectively, and does not show antimicrobial or channel modulating activities. We have concluded that the main function of magnificamide is the inhibition of &alpha, amylases, therefore, its functionally active recombinant analog is a promising agent for further studies as a potential drug candidate for the treatment of the type 2 diabetes mellitus.

Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA

Aquaporins (AQPs) are membrane water/glycerol channels with essential roles in biological systems, as well as being promising targets for therapy and imaging. Using a stopped-flow method, a series of gold(III), platinum(II) and copper(II) complexes bearing nitrogen donor ligands, such as 1,10-phenatroline, 2,2′-bipyridine, 4,4′-dimethyl-2,2′-bipyridine, 4,4′-diamino-2,2′-bipyridine and 2,2′;6′,2“-terpyridine, were evaluated in human red blood cells expressing AQP1 and AQP3, responsible for water and glycerol movement, respectively. The results showed that the gold(III) complexes selectively modulate AQP3 over AQP1. Molecular modeling and density functional theory (DFT) calculations were subsequently performed to rationalize the observations and to investigate the possible molecular mechanism through which these gold compounds act on their putative target (AQP3). In the absence of any crystallographic data, a previously reported homology model was used for this purpose. Combined, the findings of this study show that potent and selective modulation of these solute channels is possible, however further investigation is required into the selectivity of this class of agents against all AQP isoforms and their potential therapeutic uses.

Chloroplasts from land plants and algae originated from an endosymbiotic event, most likely involving an ancestral photoautotrophic prokaryote related to cyanobacteria. Both chloroplasts and cyanobacteria have thylakoid membranes, harboring pigment-protein complexes that perform the light-dependent reactions of oxygenic photosynthesis. The composition, function and regulation of these complexes have thus far been the major topics in thylakoid membrane research. For many decades, we have also accumulated biochemical and electrophysiological evidence for the existence of solute transthylakoid transport activities that affect photosynthesis. However, research dedicated to molecular identification of the responsible proteins has only recently emerged with the explosion of genomic information. Here we review the current knowledge about channels and transporters from the thylakoid membrane of Arabidopsis thaliana and of the cyanobacterium Synechocystis sp. PCC 6803. No homologues of these proteins have been characterized in algae, although similar sequences could be recognized in many of the available sequenced genomes. Based on phylogenetic analyses, we hypothesize a host origin for most of the so far identified Arabidopsis thylakoid channels and transporters. Additionally, the shift from a non-thylakoid to a thylakoid location appears to have occurred at different times for different transport proteins. We propose that closer control of and provision for the thylakoid by products of the host genome has been an ongoing process, rather than a one-step event. Some of the proteins recruited to serve in the thylakoid may have been the result of the increased specialization of its pigment-protein composition and organization in green plants. Electronic supplementary material The online version of this article (doi:10.1007/s00018-013-1412-3) contains supplementary material, which is available to authorized users.

KCNE3 modulates the KCNQ1 K+ channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state., The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium., Graphical abstract Schematic representation of Mep45 in the outer membrane of S. ruminantium. The C-terminal region of Mep45 forms a non-specific diffusion channel.

Background Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. Methods A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. Results Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F′/G′ cassette role and substrate access channels. Conclusions Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. General significance The result provides a new basis for determining the structural elements that control functions in complex enzymes.

Peroxisomes perform a large variety of metabolic functions that require a constant flow of metabolites across the membranes of these organelles. Over the last few years it has become clear that the transport machinery of the peroxisomal membrane is a unique biological entity since it includes nonselective channels conducting small solutes side by side with transporters for ‘bulky’ solutes such as ATP. Electrophysiological experiments revealed several channel-forming activities in preparations of plant, mammalian, and yeast peroxisomes and in glycosomes of Trypanosoma brucei . The properties of the first discovered peroxisomal membrane channel – mammalian Pxmp2 protein – have also been characterized. The channels are apparently involved in the formation of peroxisomal shuttle systems and in the transmembrane transfer of various water-soluble metabolites including products of peroxisomal β-oxidation. These products are processed by a large set of peroxisomal enzymes including carnitine acyltransferases, enzymes involved in the synthesis of ketone bodies, thioesterases, and others. This review discusses recent data pertaining to solute permeability and metabolite transport systems in peroxisomal membranes and also addresses mechanisms responsible for the transfer of ATP and cofactors such as an ATP transporter and nudix hydrolases. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.

Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis–Ichthyosis–Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

VDAC is now universally accepted as the channel in the mitochondrial outer membrane responsible for metabolite flux in and out of mitochondria. Its discovery occurred over two independent lines of investigation in the 1970s and 80s. This retrospective article describes the history of VDAC's discovery and how these lines merged in a collaboration by the authors. The article was written to give the reader a sense of the role played by laboratory environment, personalities, and serendipity in the discovery of the molecular basis for the unusual permeability properties of the mitochondrial outer membrane. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

International audience; Potassium (K+) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K+ in plant homeostasis was shown. It was demonstrated that K+ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K+ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K+ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K+ channel.

The influenza M2 H+ channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed Ks measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system.