Proton Pumping Function Research Articles

Contribution of Proteorhodopsin to Light-Dependent Biological Responses in Hymenobacter nivis P3T Isolated from Red Snow in Antarctica.

Proteorhodopsin (PR) is a major family of microbial rhodopsins that function as light-driven outward proton pumps. PR is now widely recognized for its ecological importance as a molecule responsible for solar energy flow in various ecosystems on the earth. However, few concrete examples of the actual use of light by natural microorganisms via PR have been demonstrated experimentally. This study reveals one example of that in a cryophilic bacterium Hymenobacter nivis P3T isolated from red snow in Antarctica. The results demonstrate light-dependent biochemical and biological responses in H. nivis cells, such as the proton pump activity of H. nivis PR (HnPR), which leads to the production of proton motive force, cellular ATP production, and cell growth. In addition, the results of this study demonstrate the photochemical properties of a PR, namely, HnPR, in the membrane of a natural host bacterium. The photocycle of HnPR was much faster than other PRs even at 5 °C, indicating that the proton pump function of HnPR has adapted to the low-temperature environment of Antarctica. Although it is well-known that PR helps natural host microorganisms to use light energy, this study provides another concrete example for understanding the biological role of PR by demonstrating the link between the molecular functions of PR and the light-dependent biochemical and biological responses of a PR-bearing host.

Effects of EGFR Inhibitors on Proton Pump Function in Gastric Mucosa and Its Mechanism

This research was carried out to detect the influence of the proton pump mechanism function on the EGFR inhibitor. Human gastric cancer cell line MKN-45 cells were divided into different concentrations of EGFR inhibitors and treated with either AG1478 or PD153035. Western Blot and immunofluorescence describe the expression of proton pump (H+/K+ ATPase). Next, the phosphorylated levels of ERK and AKT were analyzed. The overall disruption and strong results in the effects of EGFR inhibition, proton-pump transcription, and phosphorylation of ERK and AKT indicated the vital importance of this pathway. Moreover, this study also indicated that EGFR inhibitors were inhibitors of the EGFR signaling pathways themselves due to a decrease in the expression and activity of the proton pump; moreover, this inhibitory effect was closely related to drug concentration. Such findings open up new treatment opportunities for various gastric diseases and underline the possibly critical role of EGFR in gastric acid regulation.

Unique hydrogen-bonding network in a viral channelrhodopsin

Channelrhodopsins (CRs) are used as key tools in optogenetics, and novel CRs, either found from nature or engineered by mutation, have greatly contributed to the development of optogenetics. Recently CRs were discovered from viruses, and crystal structure of a viral CR, OLPVR1, reported a very similar water-containing hydrogen-bonding network near the retinal Schiff base to that of a light-driven proton-pump bacteriorhodopsin (BR). In both OLPVR1 and BR, nearly planar pentagonal cluster structures are comprised of five oxygen atoms, three oxygens from water molecules and two oxygens from the Schiff base counterions. The planar pentagonal cluster stabilizes a quadrupole, two positive charges at the Schiff base and an arginine, and two negative charges at the counterions, and thus plays important roles in light-gated channel function of OLPVR1 and light-driven proton pump function of BR. Despite similar pentagonal cluster structures, present FTIR analysis revealed different hydrogen-bonding networks between OLPVR1 and BR. The hydrogen bond between the protonated Schiff base and a water is stronger in OLPVR1 than in BR, and internal water molecules donate hydrogen bonds much weaker in OLPVR1 than in BR. In OLPVR1, the bridged water molecule between the Schiff base and counterions forms hydrogen bonds to D76 and D200 equally, while the hydrogen-bonding interaction is much stronger to D85 than to D212 in BR. The present interpretation is supported by the mutation results, where D76 and D200 equally work as the Schiff base counterions in OLPVR1, but D85 is the primary counterion in BR. This work reports highly sensitive hydrogen-bonding network in the Schiff base region, which would be closely related to each function through light-induced alterations of the network.

Membrane Proteins in Action Monitored by pH-Responsive Liquid Crystal Biosensors.

Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.

Dmxl1 Is an Essential Mammalian Gene that Is Required for V-ATPase Assembly and Function In Vivo.

The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.

The assembly factor Dmxl1 is required for H+(V)-ATPase assembly and function in mammals in vivo

The V-ATPase (proton pump) generates electrochemical gradients across cell membranes, driving essential biological processes. Critically, the two V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in serious pathologies such as cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting intercalated cells (IC) in the kidney. However, very little is known about the molecular regulation of V-ATPase in mammals. We have identified a novel interactor of the IC-enriched B1 subunit of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 protein (Dmxl1), aka Rabconnectin-3A (Rbcn3A). The yeast homologue of Dmxl1, Rav1p, is part of a protein complex that catalyzes the reversible assembly of the V1 and VO domains, thus regulating proton pumping activity. We, therefore, hypothesized that Dmxl1 is an assembly factor for the V-ATPase in mammalian cells. We generated kidney IC-specific Dmxl1 knockout (KO) mice and they had a high urine pH, similar to B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting of kidney lysates showed decreased B1 V-ATPase expression, but other V-ATPase subunits were normal. B1 (V1 domain) and a4 (VO domain) subunits were immunolocalized in ICs and apical localization quantified using FIJI. Both B1 and a4 were more intracellular in Dmxl1 KO ICs relative to control mice. A Pearson’s correlation analysis of B1 and a4 staining revealed a significant decrease in colocalization in Dmxl1 KO ICs, suggesting reduced B1/a4 assembly. In parallel, subcellular fractionation experiments revealed less V1 domain-associated B1 subunit in the membrane fraction relative to the cytosol. Furthermore, a Proximity Ligation Assay (PLA) was performed using probes against B1 and a4 to confirm decreased assembly of the two domains in KO ICs. When the two probes are within 40 μM of each other, like when the domains are assembled, this results in a fluorescent PLA punctum. As expected, we observed a significant decrease in the area of PLA puncta per cell in KO animals relative to control animals. These data demonstrate that the V-ATPase is less assembled in Dmxl1 KO ICs, relative to controls. We propose that loss of Dmxl1 reduces assembly of the V-ATPase holoenzyme, thereby inhibiting its proton pumping function. Dmxl1 may mediate recruitment of the V1 domain to the membrane and facilitate its assembly with the transmembrane VO domain. In the absence of Dmxl1, V1 is instead targeted for degradation, explaining the inability of these mice to properly acidify their urine. We conclude that Dmxl1 is a bona-fide mammalian V-ATPase assembly factor that functions similarly to its yeast homolog, Rav1p in affecting V-ATPase assembly. NIH/NIDDK R01DK121848 to DB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Covalent Bond between the Lys-255 Residue and the Main Chain Is Responsible for Stable Retinal Chromophore Binding and Sodium-Pumping Activity of Krokinobacter Rhodopsin 2.

Microbial rhodopsins are light-receptive proteins with various functions triggered by the photoisomerization of the retinal chromophore from the all-trans to 13-cis configuration. A retinal chromophore is covalently bound to a lysine residue in the middle of the seventh transmembrane helix via a protonated Schiff base. Bacteriorhodopsin (BR) variants lacking a covalent bond between the side chain of Lys-216 and the main chain formed purple pigments and exhibited a proton-pumping function. Therefore, the covalent bond linking the lysine residue and the protein backbone is not considered a prerequisite for microbial rhodopsin function. To further examine this hypothesis regarding the role of the covalent bond at the lysine side chain for rhodopsin functions, we investigated K255G and K255A variants of sodium-pumping rhodopsin, Krokinobacter rhodopsin 2 (KR2), with an alkylamine retinal Schiff base (prepared by mixing ethyl- or n-propylamine and retinal (EtSB or nPrSB)). The KR2 K255G variant incorporated nPrSB and EtSB as similarly to the BR variants, whereas the K255A variant did not incorporate these alkylamine Schiff bases. The absorption maximum of K255G + nPrSB was 524-516 nm, which was close to the 526 nm absorption maximum of the wild-type + all-trans retinal (ATR). However, the K255G + nPrSB did not exhibit any ion transport activity. Since the KR2 K255G variant easily released nPrSB during light illumination and did not form an O intermediate, we concluded that a covalent bond at Lys-255 is important for the stable binding of the retinal chromophore and formation of an O intermediate to achieve light-driven Na+ pump function in KR2.

An optoelectronic device based on bacteriorhodopsin with modulated photocurrent waveform

Developing novel photoelectric devices by functional biophotoelectric materials, especially environmentally friendly and biocompatible natural biomaterials are of great significance in the emerging field of bioelectronics. Bacteriorhodopsin (bR) has attracted much attention because of its good stability and light response since it was found. The response signal of photoelectric devices employing proton pump function of bR often exists in differential form, however, the typical electronic unit prefer to obtain square-wave signals with controllable duty cycles, which is the basis of the logic circuit structure. In this paper, we report an optoelectronic device based on bR molecule with modulated photocurrent waveform, which is achieved to use external bias to increase the duration of photocurrent, so as to form photocurrent similar to square wave signal. This improvement will contribute to the application of such biological devices as logic units in the field of digital circuits and optical communication, and provide a new idea for the development of new optoelectronic molecular devices.

A Light-Driven Integrated Bio-Capacitor with Single Nano-Channel Modulation

Bioelectronics, an emerging discipline formed by the biology and electronic information disciplines, has maintained a state of rapid development since its birth. Amongst the various functional bioelectronics materials, bacteriorhodopsin (bR), with its directional proton pump function and favorable structural stability properties, has drawn wide attention. The main contents of the paper are as follows: Inspired by the capacitive properties of natural protoplast cell membranes, a new bio-capacitor based on bR and artificial nanochannels was constructed. As a point of innovation, microfluidic chips were integrated into our device as an ion transport channel, which made the bio-capacitor more stable. Meanwhile, a single nanopore structure was integrated to improve the accuracy of the device structure. Experiments observed that the size of the nanopore affected the ion transmission rate. Consequently, by making the single nanopore’s size change, the photocurrent duration time (PDT) of bR was effectively regulated. By using this specific phenomenon, the original transient photocurrent was successfully transformed into a square-like wave.

Identification of ATP6V1C2 as a novel candidate gene for distal tubular acidosis

Young onset distal tubular acidosis is a rare genetic disorder that can lead, if untreated, to many complications. Mutations in few genes account for almost half of the cases, whereas the molecular mechanisms accounting for the remaining cases are still unknown. The present study reports the use of whole-exome sequencing to identify new dRTA-causing genes and demonstrates that inactivating mutations in the ATP6V1C2 gene impair renal proton pump function.

How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?

We often encounter a case where two proteins, whose amino-acid sequences are similar, are quite different with regard to the thermostability. As a striking example, we consider the two seven-transmembrane proteins: recently discovered Rubrobacter xylanophilus rhodopsin (RxR) and long-known bacteriorhodopsin from Halobacterium salinarum (HsBR). They commonly function as a light-driven proton pump across the membrane. Though their sequence similarity and identity are ∼71 and ∼45%, respectively, RxR is much more thermostable than HsBR. In this study, we solve the three-dimensional structure of RxR using X-ray crystallography and find that the backbone structures of RxR and HsBR are surprisingly similar to each other: The root-mean-square deviation for the two structures calculated using the backbone Cα atoms of the seven helices is only 0.86 Å, which makes the large stability difference more puzzling. We calculate the thermostability measure and its energetic and entropic components for RxR and HsBR using our recently developed statistical-mechanical theory. The same type of calculation is independently performed for the portions playing essential roles in the proton-pumping function, helices 3 and 7, and their structural properties are related to the probable roles of water molecules in the proton-transporting mechanism. We succeed in elucidating how RxR realizes its exceptionally high stability with the original function being retained. This study provides an important first step toward the establishment of a method correlating microscopic, geometric characteristics of a protein with its thermodynamic properties and enhancing the thermostability through amino-acid mutations without vitiating the original function.

Oriented bacteriorhodopsin/polyaniline hybrid bio-nanofilms as photo-assisted electrodes for high performance supercapacitors

Bacteriorhodopsin was used to improve the capacitive performance of PANI supercapacitors by its proton pumping and photoelectric function.

X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity

Cytochrome c oxidase (CcO) is the terminal oxidase of cellular respiration, reducing O2 to water and pumping protons. X-ray structural features have suggested that CcO pumps protons via a mechanism involving electrostatic repulsions between pumping protons in the hydrogen-bond network of a proton-conducting pathway (the H-pathway) and net positive charges created upon oxidation of an iron site, heme a (Fea2+), for reduction of O2 at another iron site, heme a3 (Fea32+). The protons for pumping are transferred to the hydrogen-bond network from the N-side via the water channel of the H-pathway. Back-leakage of protons to the N-side is thought to be blocked by closure of the water channel. To experimentally test this, we examined X-ray structures of the azide-bound, oxidized bovine CcO and found that an azide derivative (N3−–Fea33+, CuB2+–N3−) induces a translational movement of the heme a3 plane. This was accompanied by opening of the water channel, revealing that Fea3 and the H-pathway are tightly coupled. The channel opening in the oxidized state is likely to induce back-leakage of pumping protons, which lowers the proton level in the hydrogen-bond network during enzymatic turnover. The proton level decrease weakens the electron affinity of Fea, if Fea electrostatically interacts with protons in the hydrogen-bond network. The previously reported azide-induced redox-potential decrease in Fea supports existence of the electrostatic interaction. In summary, our results indicate that the H-pathway is critical for CcO's proton-pumping function.

Valproate inhibits glucose-stimulated insulin secretion in beta cells.

Valproate (VPA), an FDA approved anti-epileptic drug with a half-life of 12-18h in humans, has been shown to perturb the vacuolar proton pump (vH+-ATPase) function in yeasts by inhibiting myo-inositol phosphate synthase, the first and rate-limiting enzyme in inositol biosynthesis, thereby resulting in inositol depletion. vH+-ATPase transfers protons (H+) across cell membranes, which help maintain pH gradients within cells necessary for various cellular functions including secretion. This proton pump has a membrane (V0) and a soluble cytosolic (V1) domain, with C-subunit associated with V1. In secretory cells such as neurons and insulin-secreting beta cells, vH+-ATPase acidifies vesicles essential for secretion. In this study, we demonstrate that exposure of insulin-secreting Min6 cells to a clinical dose of VPA results in inositol depletion and loss of co-localization of subunit C of vH+-ATPase with insulin-secreting granules. Consequently, a reduction of glucose-stimulated insulin secretion is observed following VPA exposure. These results merit caution and the reassessment of the clinical use of VPA.

High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

Summary The membrane‐bound proton‐pumping pyrophosphatase (V‐PPase), together with the V‐type H+‐ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V‐PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton‐pumping functions has yet to be dissected.For a better understanding of the molecular processes underlying V‐PPase‐dependent salt tolerance, we transiently overexpressed the pyrophosphate‐driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch‐clamp, impalement electrodes and pH imaging.NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt‐untreated conditions, V‐PPase‐overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP‐hyperactive cells from cell death. Furthermore, a salt‐induced rise in V‐PPase but not of V‐ATPase pump currents was detected in nontransformed plants.The results indicate that under normal growth conditions, plants need to regulate the V‐PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V‐PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton‐coupled Na+ sequestration.

A Review on Bacteriorhodopsin-Based Bioelectronic Devices.

Bacteriorhodopsin protein extracted from Halobacterium salinarum is widely used in many biohybrid electronic devices and forms a research subject known as bioelectronics, which merges biology with electronic technique. The specific molecule structure and components of bR lead to its unique photocycle characteristic, which consists of several intermediates (bR, K, L, M, N, and O) and results in proton pump function. In this review, working principles and properties of bacteriorhodopsin are briefly introduced, as well as bR layer preparation method. After that, different bR-based devices divided into photochemical and photoelectric applications are shown. Finally, outlook and conclusions are drawn to inspire new design of high-performance bR-based biohybrid electronic devices.

Breaking up and making up: The secret life of the vacuolar H+ -ATPase.

The vacuolar ATPase (V-ATPase; V1 Vo -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Functional Reconstitution of V-ATPase Proteolipid Ring into a Planar Lipid Membrane

In this work, we show the first purification and functional characterization of the proteolipid ring (c-ring) complex of the membrane-bound Vo sector of the eukaryotic vacuolar H+-ATPase (V-ATPase) from the yeast Saccharomyces cerevisiae using techniques of membrane biochemistry, structural biology, and single-molecule electrophysiology. Our work was motivated by, and builds on, a large body of literature spanning more than three decades that suggested that Vo, besides its proton pumping function in holo V-ATPase, has other, so-called “non-canonical” functions in essential processes, such as membrane fusion and neurotransmitter release. However, direct evidence that Vo, and specifically, its core proteolipid ring (c-ring) complex, possesses the required properties to fulfill these non-canonical functions is still lacking. Our results presented here show that the c-ring forms a large-conductance transmembrane pore, which inserted into planar lipid bilayers with a preferred orientation. Moreover, using single-particle cryo-electron microscopy, we demonstrate that the purified c-ring forms dimers through an interaction mediated by the cytoplasmic loops of the c subunits. Taken together, these findings provide the first direct attestation in support of the proposed non-canonical functions of the V-ATPase Vo sector.1 This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.). 1. Couoh-Cardel, S.; Hsueh, Y. C.; Wilkens, S.; Movileanu, L., Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore. Scientific reports 2016, 6, 24774.

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study.

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

Development of UV Light Irradiation Patterning of Bacteriorhodopsin Thin Films for Biomimetic Functional Devices.

We developed a new patterning method for bacteriorhodopsin (bR) thin films using UV light irradiation. The proton pump function of bR thin films can be deactivated with UV light irradiation. Inactivation of the proton pump function of bR is related to structural changes or photo-bleaching of the retinal in bR using UV light exposure, which was confirmed with absorption and Raman spectroscopy measurements. Utilizing inactivation of the proton pump function with UV light irradiation, we prepared a bR photocell with a stripe-patterned bR thin film and measured its photocurrent response. The new patterning method is applicable to complicated patterning and patterning with a higher spatial resolution, which extends the application of bR thin films as sensor devices.