Taxifolin and gastro-adhesive microparticles containing taxifolin promotes gastric healing in vivo, inhibits Helicobacter pylori in vitro and proton pump reversibly in silico.

Taxifolin (3,5,7,3,4-pentahydroxy flavanone or dihydroquercetin, Tax) was identified as a gastroprotective compound and a gastroadhesive formulation was recently developed to prolong its residence time and release in the stomach. So, the gastric healing effectiveness of Tax and gastro-mucoadhesive microparticles containing Tax (MPTax) against the acetic acid induced-gastric ulcer in rats was investigated in this study. Moreover, the interactions between Tax and H+/K+-ATPase were investigated in silico, and its anti- H. pylori activity was determined in vitro. The oral treatment with MPTax (81.37 mg/kg, containing 12.29% of Tax) twice a day for seven days reduced the ulcer area by 63%, compared to vehicle-treated group (Veh: 91.9 ± 10.3 mm2). Tax (10 mg/kg, p.o) reduced the ulcer by 40% but with a p = 0.07 versus Veh group. Histological analysis confirmed these effects. Tax and MPTax increased the gastric mucin amount, reduced the myeloperoxidase activity, and increased the glutathione reduced content at ulcer site. However, only MPTax decreased the lipoperoxide accumulation at ulcer site. Besides, Tax and MPTax normalize the catalase and glutathione S-transferase activity. Tax showed reversible interaction with H+/K+-ATPase in silico and its anti-H. pylori effects was confirmed (MIC = 625 µg/mL). These results suggest that the antiulcer property of Tax involves the strengthening of the gastric protective factors in parallel to its inhibitory interaction with H+/K+-ATPase and H. pylori. Considering that ulcer healing action displayed by Tax was favored by gastroadhesive microparticles, this approach seems to be promising for its oral delivery to treat acid-peptic diseases.

Channelrhodopsin-mediated optogenetics highlights a central role of depolarization-dependent plant proton pumps.

In plants, environmental stressors trigger plasma membrane depolarizations. Being electrically interconnected via plasmodesmata, proper functional dissection of electrical signaling by electrophysiology is basically impossible. The green alga Chlamydomonas reinhardtii evolved blue light-excited channelrhodopsins (ChR1, 2) to navigate. When expressed in excitable nerve and muscle cells, ChRs can be used to control the membrane potential via illumination. In Arabidopsis plants, we used the algal ChR2-light switches as tools to stimulate plasmodesmata-interconnected photosynthetic cell networks by blue light and monitor the subsequent plasma membrane electrical responses. Blue-dependent stimulations of ChR2 expressing mesophyll cells, resting around -160 to -180 mV, reproducibly depolarized the membrane potential by 95 mV on average. Following excitation, mesophyll cells recovered their prestimulus potential not without transiently passing a hyperpolarization state. By combining optogenetics with voltage-sensing microelectrodes, we demonstrate that plant plasma membrane AHA-type H+-ATPase governs the gross repolarization process. AHA2 protein biochemistry and functional expression analysis in Xenopus oocytes indicates that the capacity of this H+ pump to recharge the membrane potential is rooted in its voltage- and pH-dependent functional anatomy. Thus, ChR2 optogenetics appears well suited to noninvasively expose plant cells to signal specific depolarization signatures. From the responses we learn about the molecular processes, plants employ to channel stress-associated membrane excitations into physiological responses.

A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl-/H+ transport cycle.

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl- transport that reconciles existing data on all CLC-type proteins.

Cells are shielded from harmful molecules and other threats by a thin, flexible layer called the membrane. However, this barrier also prevents chloride, sodium, protons and other ions from moving in or out of the cell. Channels and transporters are two types of membrane proteins that form passageways for these charged particles. Channels let ions flow freely from one side of the membrane to the other. To do so, these proteins change their three-dimensional shape to open or close as needed. On the other hand, transporters actively pump ions across the membrane to allow the charged particles to accumulate on one side. The shape changes needed for that type of movement are different: the transporters have to open a passageway on one side of the membrane while closing it on the other side, alternating openings to one side or the other. In general, channels and transporters are not related to each other, but one exception is a group called CLCs proteins. Present in many organisms, this family contains a mixture of channels and transporters. For example, humans have nine CLC proteins: four are channels that allow chloride ions in and out, and five are 'exchange transporters' that make protons and chloride ions cross the membrane in opposite directions. These proteins let one type of charged particle move freely across the membrane, which generates energy that the transporter then uses to actively pump the other ion in the direction needed by the cell. Yet, the exact three-dimensional changes required for CLC transporters and channels to perform their roles are still unknown. To investigate this question, Chavan, Cheng et al. harnessed a technique called X-ray crystallography, which allows scientists to look at biological molecules at the level of the atom. This was paired with other methods to examine a CLC mutant that adopts the shape of a normal CLC transporter when it is loaded with a proton. The experiments revealed how various elements in the transporter move relative to each other to adopt a structure that allows protons and chloride ions to enter the protein from opposite sides of the membrane, using separate pathways. While obtained on a bacterial CLC, these results can be applied to other CLC channels and transporters (including those in humans), shedding light on how this family transports charged particles across membranes. From bone diseases to certain types of seizures, many human conditions are associated with poorly functioning CLCs. Understanding the way these structures change their shapes to perform their roles could help to design new therapies for these health problems.

A calibrated optogenetic toolbox of stable zebrafish opsin lines.

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

[Negative regulation of gastric proton pump by desialylation suggested by fluorescent imaging with the sialic acid-specific nanoprobe].

Gastric proton pump (H+,K+-ATPase) which is responsible for H+ secretion of gastric acid (HCl) in gastric parietal cells is the major therapeutic target for treatment of acid-related diseases. H+,K+-ATPase consists of two subunits, a catalytic α-subunit (αHK) and a glycosylated ß-subunit (ßHK). N-glycosylation of ßHK is essential for trafficking and stability of αHK in apical membrane of gastric parietal cells. Terminal sialic acid residues on sugar chains have an important role in various cellular functions. Recently, we succeeded in visualizing the sialylation and desialylation dynamics of ßHK using a fluorescence bioimaging nanoprobe consisting of biocompatible polymers conjugated with lectins for detecting sialic acid. In H+,K+-ATPase-expressing cell lines, rat gastric mucosa, and primary culture of rat gastric parietal cells, fluorescence imaging of sialic acid with the nanoprobe showed that sialylation of ßHK is regulated by intragastric pH and that inhibition of gastric acid secretion induces desialylation of ßHK. In biochemical and pharmacological studies, we revealed that enzyme activity of αHK is negatively regulated by desialylation of ßHK. Our studies uncovered a novel negative-feedback mechanism of H+,K+-ATPase in which sialic acids of ßHK positively regulates H+,K+-ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of ßHK. In this topic, we introduce the overview of our research using the bioimaging nanoprobe.

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy.

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.

The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H+ gradient to synthesize pyrophosphate.

Energy partitioning and plant growth are mediated in part by a type I H+-pumping pyrophosphatase (H+-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H+-PPase from Arabidopsis also functions in "reverse mode" to synthesize PPi using the transmembrane H+ gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon Pi application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H+-PPase mutant lines (vhp1-1 and fugu5-1) lacking the classical PPi-induced outward currents related to H+ pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H+-PPase. Current amplitudes related to reverse-mode H+ transport depended on the membrane potential, cytosolic Pi concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H+-PPase (AVP1) demonstrated Pi-dependent PPi synthase activity in the presence of a pH gradient. Our work establishes that a plant H+-PPase can operate as a PPi synthase beyond its canonical role in vacuolar acidification and cytosolic PPi scavenging. We propose that the PPi synthase activity of H+-PPase contributes to a cascade of events that energize plant growth.

The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping.

Under anaerobic conditions, Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein (HybB). To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of Hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In the present paper, we describe a new overexpression system that has facilitated the determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.

Role of Proton Pumps in Tumorigenesis.

One of the differences between normal and cancer cells is lower pH of the extracellular space in tumors. Low pH in the extracellular space activates proteases and stimulates tumor invasion and metastasis. Tumor cells display higher level of the HIF1α transcription factor that promotes cell switch from mitochondrial respiration to glycolysis. The terminal product of glycolysis is lactate. Lactate formation from pyruvate is catalyzed by the specific HIF1α-dependent isoform of lactate dehydrogenase A. Because lactate accumulation is deleterious for the cell, it is actively exported by monocarboxylate transporters. Lactate is cotransported with proton, which acidifies the extracellular space. Another protein that contributes to proton concentration increase in the extracellular space is tumor-specific HIF1α-dependent carbonic anhydrase IX, which generates a proton in the reaction between carbon dioxide and water. The activity of Na+/H+ exchanger (another protein pump) is stimulated by stress factors (e.g. osmotic shock) and proliferation stimuli. This review describes the mechanisms of proton pump activation and reviews results of studies on effects of various proton pump inhibitors on tumor functioning and growth in cell culture and in vivo. The prospects of combined application of proton pump inhibitors and cytostatics in cancer therapy are discussed.

A natural light-driven inward proton pump.

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.

Functional and Photochemical Characterization of a Light-Driven Proton Pump from the Gammaproteobacterium Pantoea vagans.

Photoactive retinal proteins are widely distributed throughout the domains of the microbial world (i.e., bacteria, archaea, and eukarya). Here we describe three retinal proteins belonging to a phylogenetic clade with a unique DTG motif. Light-induced decrease in the environmental pH and its inhibition by carbonyl cyanide m-chlorophenylhydrazone revealed that these retinal proteins function as light-driven outward electrogenic proton pumps. We further characterized one of these proteins, Pantoea vagans rhodopsin (PvR), spectroscopically. Visible spectroscopy and high-performance liquid chromatography revealed that PvR has an absorption maximum at 538 nm with the retinal chromophore predominantly in the all-trans form (>90%) under both dark and light conditions. We estimated the pKa values of the protonated Schiff base of the retinal chromophore and its counterion as approximately 13.5 and 2.1, respectively, by using pH titration experiments, and the photochemical reaction cycle of PvR was measured by time-resolved flash-photolysis in the millisecond timeframe. We observed a blue-shifted and a red-shifted intermediate, which we assigned as M-like and O-like intermediates, respectively. Decay of the M-like intermediate was highly sensitive to environmental pH, suggesting that proton uptake is coupled to decay of the M-like intermediate. From these results, we propose a putative model for the photoreaction of PvR.

Redox-induced activation of the proton pump in the respiratory complex I.

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.

Nitric oxide contributes to minerals absorption, proton pumps and hormone equilibrium under cadmium excess in Trifolium repens L. plants.

Nitric oxide (NO) is a stress-signaling molecule in plants that mediates a wide range of physiological processes and responses to metal toxicity. In this work, various NO modulators (NO donor: SNP; NO scavenger: cPTIO; NO synthase inhibitor: l-NAME; and SNP analogs: sodium nitrite/nitrate and sodium ferrocyanide) were investigated to determine the role of NO in Trifolium repens L. plants exposed to Cd. Cd (100µM) markedly reduced biomass, NO production and chlorophyll (Chl a, Chl b and total Chl) concentration but stimulated reactive oxygen species (ROS) and Cd accumulation in plants. SNP (50µM) substantially attenuated growth inhibition, reduced hydrogen peroxide (H2O2) and malonyldialdehyde (MDA) levels, stimulated ROS-scavenging enzymes/agents, and mitigated the H(+)-ATPase inhibition in proton pumps. Interestingly, SNP considerably up-regulated the levels of jasmonic acid (JA) and proline in plant tissues but down-regulated the levels of ethylene (ET) in both shoots and roots and the level of salicylic acid (SA) in roots only, which might be related to the elevated NO synthesis. Additionally, SNP (25-200µM) regulated mineral absorption and, particularly at 50µM, significantly enhanced the uptake of shoot magnesium (Mg) and copper (Cu) and of root calcium (Ca), Mg and iron (Fe). Nevertheless, the effects of SNP on plant growth were reversed by cPTIO and l-NAME, suggesting that the protective effect of SNP might be associated with NO synthesis in vivo. Moreover, SNP analogs did not display roles similar to that of SNP. These results indicated that NO depleted Cd toxicity by eliminating oxidative damage, enhancing minerals absorption, regulating proton pumps, and maintaining hormone equilibrium.

A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration.

We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H(+)-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi.

Voltage dependence of proton pumping by bacteriorhodopsin mutants with altered lifetime of the M intermediate.

The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H(+)] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H(+) pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H(+) pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H(+) pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H(+) gradient generation.

The cystic fibrosis of exocrine pancreas.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is highly expressed in the pancreatic duct epithelia and permits anions and water to enter the ductal lumen. This results in an increased volume of alkaline fluid allowing the highly concentrated proteins secreted by the acinar cells to remain in a soluble state. This work will expound on the pathophysiology and pathology caused by the malfunctioning CFTR protein with special reference to ion transport and acid-base abnormalities both in humans and animal models. We will also discuss the relationship between cystic fibrosis (CF) and pancreatitis, and outline present and potential therapeutic approaches in CF treatment relevant to the pancreas.

Renal intercalated cells are rather energized by a proton than a sodium pump.

The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na(+) has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na(+)/K(+)-ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H(+) vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na(+)/K(+)-ATPase had no effects. Basolateral NaCl exit from ß-intercalated cells was independent of the Na(+)/K(+)-ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H(+) V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H(+) V-ATPase.

A conserved asparagine in a P-type proton pump is required for efficient gating of protons.

The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H(+)-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pKa of the aspartate. However, other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H(+)-ATPases. In the crystal structure of the plant plasma membrane H(+)-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Sensitivity toward orthovanadate was increased when Asn-106 was substituted with an aspartate residue, but decreased in mutants with alanine, lysine, glutamine, or threonine replacement of Asn-106. The apparent proton affinity was decreased for all mutants, most likely due to a perturbation of the local environment of Asp-684. Altogether, our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane.

Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping.

Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.

Acidification of the synaptic cleft of cone photoreceptor terminal controls the amount of transmitter release, thereby forming the receptive field surround in the vertebrate retina.

In the vertebrate retina, feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the formation of the center-surround receptive field of retinal cells, which induces contrast enhancement of visual images. The mechanism underlying surround inhibition is not fully understood. In this review, we discuss this issue, focusing on our recent hypothesis that acidification of the synaptic cleft of the cone photoreceptor terminal causes this inhibition by modulating the Ca channel of the terminals. We present evidence that the acidification is caused by proton excretion from HCs by a vacuolar type H(+) pump. Recent publications supporting or opposing our hypothesis are discussed.