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Recent development of optogenetic techniques, which utilize light-driven ion channels or ion pumps for controlling the activity of excitable cells, has greatly facilitated the investigation of nervous systems in vivo. A new generation of optical silencers includes outward-directed proton pumps, such as Arch, which have several advantages over currently widely used halorhodopsin (NpHR). These advantages include the resistance to inactivation during prolonged illumination and the ability to generate a larger optical current from low intensity light. C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses. In this article, we will outline the practical aspects of using of Arch and other proton pumps as optogenetic tools in C. elegans.
One major objective of synthetic biology is the bottom-up assembly of minimalistic nanocells consisting of lipid or polymer vesicles as architectural scaffolds and of membrane and soluble proteins as functional elements. However, there is no reliable method to orient membrane proteins reconstituted into vesicles. Here, we introduce a simple approach to orient the insertion of the light-driven proton pump proteorhodopsin (PR) into liposomes. To this end, we engineered red or green fluorescent proteins to the N- or C-terminus of PR, respectively. The fluorescent proteins optically identified the PR constructs and guided the insertion of PR into liposomes with the unoccupied terminal end facing inward. Using the PR constructs, we generated proton gradients across the vesicle membrane along predefined directions such as are required to power (bio)chemical processes in nanocells. Our approach may be adapted to direct the insertion of other membrane proteins into vesicles.
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2ND3. We conclude that movement of loop TMH1-2ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.
Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.
Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.
A single Na(+)/K(+)-ATPase pumps three Na(+) outwards and two K(+) inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na(+) than K(+) generates outward current across the cell membrane. Less well understood is the ability of Na(+)/K(+) pumps to generate an inward current of protons. Originally noted in pumps deprived of external K(+) and Na(+) ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K(+) and Na(+) concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na(+) release from phosphorylated Na(+)/K(+) pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na(+)/K(+) pumps that enables proton import is not required for completion of the 3 Na(+)/2 K(+) transport cycle. However, the back-step occurs readily during Na(+)/K(+) transport when external K(+) ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na(+)-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na(+) and K(+) ions that passes through binding site II. The inferred occurrence of Na(+)/K(+) exchange and H(+) import during the same conformational cycle of a single molecule identifies the Na(+)/K(+) pump as a hybrid transporter. Whether Na(+)/K(+) pump-mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.
Pregnancy brings about strong cravings for nonfood materials, the gestational toxicities of which are not yet ascertained. In this study, we used rat models to investigate the effect of clay beverage consumption during early and late gestation on p-Type ATPases, nephrocardiac integrity, the antioxidant system, and on the activities of the renin⁻angiotensin⁻aldosterone system (RAAS). The rats at early (7th day) and late gestation (20th) were administered single doses (500 mg/kg body weight of clay beverage and examined using ELISA and spectrophotometry. The gestational clay beverage intake significantly elevated the renal hemodynamics, glomerular filtration rate (GFR), anion gap, urinary output, and blood urea nitrogen⁻creatinine ratio (BUN/Crt). At early and late gestation, clay beverage consumption elevated the heartbeat, atherogenic index of plasma, cardiac risk ratio, and atherogenic coefficients. Creatinine kinase and troponin levels after clay beverage consumption significantly increased with gestation age, while lactate dehydrogenase elevation was independent of gestation age. Mg2+-ATPase and Na⁺/K⁺-ATPase significantly decreased during gestation and were further altered with clay beverage intake. The rats showed higher RAAS activities during early and late gestation stages but greatly decreased activities after clay beverage administration. When F2-isoprostane and malondialdehyde levels were measured, slight elevations were found during pregnancy and were greatly elevated with clay beverage intake, while the glutathione reductase, catalase, and superoxide dismutase levels were decreased. We thus discourage clay beverage consumption throughout the entire pregnancy period because of these profound homeostatic imbalances and organ toxicities associated with its consumption.
In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.
In cancer cells in vitro, the glycolytic pathway and the mitochondrial tricarboxylic acid (TCA) cycle are programmed to produce more precursor molecules, and relatively less ATP, than in differentiated cells. We address the questions of whether and where these changes occur in vivo in glioblastomas grown from C6 cells in rat brain. These gliomas show some spatial organization, notably in the upregulation of membrane proton transporters near the rim.
Cytochrome c oxidase (CcO) reduces O2 in the O2-reduction site by sequential four-electron donations through the low-potential metal sites (CuA and Fea). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp51, Arg438, Glu198, the hydroxyfarnesyl ethyl group of heme a, and Ser382, respectively. These sites interact with the putative proton-pumping H-pathway. However, the metal sites responsible for each structural change have not been identified, since these changes were detected as structural differences between the fully reduced and fully oxidized CcOs. Thus, the roles of these structural changes in the CcO function are yet to be revealed. X-ray crystal structures of cyanide-bound CcOs under various oxidation states showed that the O2-reduction site controlled only the Ser382-including site, while the low-potential metal sites induced the other changes. This finding indicates that these low-potential site-inducible structural changes are triggered by sequential electron-extraction from the low-potential sites by the O2-reduction site and that each structural change is insensitive to the oxidation and ligand-binding states of the O2-reduction site. Because the proton/electron coupling efficiency is constant (1:1), regardless of the reaction progress in the O2-reduction site, the structural changes induced by the low-potential sites are assignable to those critically involved in the proton pumping, suggesting that the H-pathway, facilitating these low-potential site-inducible structural changes, pumps protons. Furthermore, a cyanide-bound CcO structure suggests that a hypoxia-inducible activator, Higd1a, activates the O2-reduction site without influencing the electron transfer mechanism through the low-potential sites, kinetically confirming that the low-potential sites facilitate proton pump.
Ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like pH gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. Such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. Using Drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. We, therefore, investigated the stage-specific distribution patterns of V-ATPase components in relation to gap-junction proteins.
Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubacteria, a large number of other eubacterial rhodopsins with diverse structures and functions have been characterized. Here, we review the body of knowledge accumulated on the four major groups of eubacterial rhodopsins, with the focus on their biophysical characterization. We discuss advances and controversies on the unique eubacterial sensory rhodopsins (as represented by Anabaena sensory rhodopsin), proton-pumping proteorhodopsins and xanthorhodopsins, as well as novel non-proton ion pumps. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
P-class pumps are specific ion transporters involved in maintaining intracellular/extracellular ion homeostasis, gene transcription, and cell proliferation and migration in all eukaryotic cells. The present review aimed to evaluate the role of P-type pumps [Na+/K+ ATPase (NKA), H+/K+ ATPase (HKA) and Ca2+-ATPase] in cancer cells across three fronts, namely structure, function and genetic expression. It has been shown that administration of specific P-class pumps inhibitors can have different effects by: i) Altering pump function; ii) inhibiting cell proliferation; iii) inducing apoptosis; iv) modifying metabolic pathways; and v) induce sensitivity to chemotherapy and lead to antitumor effects. For example, the NKA β2 subunit can be downregulated by gemcitabine, resulting in increased apoptosis of cancer cells. The sarcoendoplasmic reticulum calcium ATPase can be inhibited by thapsigargin resulting in decreased prostate tumor volume, whereas the HKA α subunit can be affected by proton pump inhibitors in gastric cancer cell lines, inducing apoptosis. In conclusion, the present review highlighted the central role of P-class pumps and their possible use and role as anticancer cellular targets for novel therapeutic chemical agents.
Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.
Many H+-pump rhodopsins conserve "H+ donor" residues in cytoplasmic (CP) half channels to quickly transport H+ from the CP medium to Schiff bases at the center of these proteins. For conventional H+ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exiguobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H+ to the Schiff bases and then capture the other H+ from the medium, whereas the Lys donor first captures H+ from the medium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer reactions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H+ donor. In contrast, for Glu-type proteorhodopsin (PR) and ESR, the embedded residues functioned well as H+ donors. These differences were further examined by focusing on the activation volumes during the H+-transfer reactions. The results revealed essential differences between archaeal H+ pump (DR) and eubacterial H+ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H+-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.
Light-driven outward H+ pumps are widely distributed in nature, converting sunlight energy into proton motive force. Here we report the characterization of an oppositely directed H+ pump with a similar architecture to outward pumps. A deep-ocean marine bacterium, Parvularcula oceani, contains three rhodopsins, one of which functions as a light-driven inward H+ pump when expressed in Escherichia coli and mouse neural cells. Detailed mechanistic analyses of the purified proteins reveal that small differences in the interactions established at the active centre determine the direction of primary H+ transfer. Outward H+ pumps establish strong electrostatic interactions between the primary H+ donor and the extracellular acceptor. In the inward H+ pump these electrostatic interactions are weaker, inducing a more relaxed chromophore structure that leads to the long-distance transfer of H+ to the cytoplasmic side. These results demonstrate an elaborate molecular design to control the direction of H+ transfers in proteins.
Wzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx from Pseudomonas aeruginosa PAO1 (WzxPa) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of WzxPa following its purification and reconstitution in phospholipid liposomes. WzxPa was capable of mediating anion flux, consistent with its cationic interior. This flux was electrogenic and modified by extraliposomal pH. Mutation of the above-mentioned acidic residues (E61, D269, and D359) reduced proton (H(+))-modified anion flux, showing the role of these amino acid side chains in H(+)-dependent transport. Wzx also mediated acidification of the proteoliposome interior in the presence of an outward anion gradient. These results indicate H(+)-dependent gating and H(+) uptake by WzxPa and allow for the first H(+)-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM. IMPORTANCE Many bacterial cell surface polysaccharides that are important for survival and virulence are synthesized at the periplasmic leaflet of the inner membrane (IM) using precursors produced in the cytoplasm. Wzx flippases are responsible for translocation of lipid-linked sugar repeat units across the IM and had been previously suggested to simply facilitate passive substrate diffusion. Through our characterization of purified Wzx in a reconstitution system described herein, we have observed protein-dependent intrinsic transport producing a change in the electrical potential of the system, with H(+) identified as the coupling ion. These results provide the first evidence for coupled (i.e., secondary active) transport by these proteins and, in conjunction with structural data, allow for an antiport mechanism to be proposed for the directed transport of lipid-linked sugar substrates across the IM. These findings bring our understanding of lipid-linked polysaccharide transporter proteins more in line with the efflux pumps to which they are evolutionarily related.
Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the β-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.
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