ATP-driven Pump Research Articles

W546 stacking disruption traps the human porphyrin transporter ABCB6 in an outward-facing transient state

Human ATP-binding cassette transporter subfamily B6 (ABCB6) is a mitochondrial ATP-driven pump that translocates porphyrins from the cytoplasm into mitochondria for heme biosynthesis. Within the transport pathway, a conserved aromatic residue W546 located in each monomer plays a pivotal role in stabilizing the occluded conformation via π-stacking interactions. Herein, we employed cryo-electron microscopy to investigate the structural consequences of a single W546A mutation in ABCB6, both in detergent micelles and nanodiscs. The results demonstrate that the W546A mutation alters the conformational dynamics of detergent-purified ABCB6, leading to entrapment of the transporter in an outward-facing transient state. However, in the nanodisc system, we observed a direct interaction between the transporter and a phospholipid molecule that compensates for the absence of the W546 residue, thereby facilitating the normal conformational transition of the transporter toward the occluded state following ATP hydrolysis. The findings also reveal that adoption of the outward-facing conformation causes charge repulsion between ABCB6 and the bound substrate, and rearrangement of key interacting residues at the substrate-binding site. Consequently, the affinity for the substrate is significantly reduced, facilitating its release from the transporter.

Advances in Intracellular Calcium Signaling Reveal Untapped Targets for Cancer Therapy.

Intracellular Ca2+ distribution is a tightly regulated process. Numerous Ca2+ chelating, storage, and transport mechanisms are required to maintain normal cellular physiology. Ca2+-binding proteins, mainly calmodulin and calbindins, sequester free intracellular Ca2+ ions and apportion or transport them to signaling hubs needing the cations. Ca2+ channels, ATP-driven pumps, and exchangers assist the binding proteins in transferring the ions to and from appropriate cellular compartments. Some, such as the endoplasmic reticulum, mitochondria, and lysosomes, act as Ca2+ repositories. Cellular Ca2+ homeostasis is inefficient without the active contribution of these organelles. Moreover, certain key cellular processes also rely on inter-organellar Ca2+ signaling. This review attempts to encapsulate the structure, function, and regulation of major intracellular Ca2+ buffers, sensors, channels, and signaling molecules before highlighting how cancer cells manipulate them to survive and thrive. The spotlight is then shifted to the slow pace of translating such research findings into anticancer therapeutics. We use the PubMed database to highlight current clinical studies that target intracellular Ca2+ signaling. Drug repurposing and improving the delivery of small molecule therapeutics are further discussed as promising strategies for speeding therapeutic development in this area.

Multipurpose Na+ ions mediate excitation and cellular homeostasis: Evolution of the concept of Na+ pumps and Na+/Ca2+ exchangers

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na+ fluxes and spatio-temporal fluctuations of Na+ concentration in cellular organelles and cellular compartments contribute to the most fundamental cellular processes such as membrane excitability and energy production. At the very core of ionic signalling lies the Na+-K+ ATP-driven pump (or NKA) which creates trans-plasmalemmal ion gradients that sustain ionic fluxes through ion channels and numerous Na+-dependent transporters that maintain cellular and tissue homeostasis. Here we present a brief account of the history of research into NKA, Na+ -dependent transporters and Na+ signalling.

Cryo-EM Structures Reveal Bilayer Remodeling during Ca2+ Activation of a TMEM16 Scramblase

The plasma membranes of eukaryotic cells are organized asymmetrically for cellular function. At rest, polar and charged lipids are sequestered to the inner leaflet by the activity of ATP-driven pumps. Activation of a specialized class of membrane proteins – phospholipid scramblases – causes rapid collapse of this asymmetry and externalization of negatively charged phosphatidylserine molecules. As a result, extracellular signaling networks, controlling processes such as apoptosis, blood coagulation, membrane fusion and repair, are activated. The TMEM16 family of membrane proteins includes phospholipid scramblases and Cl- channels, all of which are Ca2+-dependent. Prior structural and functional analyses of the fungal TMEM16 scramblase from Nectria haematococca identified a membrane-exposed hydrophilic groove that serves as the lipid translocation pathway. In the TMEM16A channel, this pathway is sealed from the membrane, thus preventing lipid access and enabling ion permeation. However, the mechanisms underlying Ca2+-dependent gating of TMEM16 scramblases, and their effects on the surrounding lipid bilayer, remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. Differences between the Ca2+-free and Ca2+-bound states reveal that Ca2+ binding induces a global rearrangement of the transmembrane and cytosolic regions, which causes the lipid pathway to open. Further, comparison of these structures and a third in the presence of a long-chain inhibitory lipid, together with functional experiments, reveal that scramblases cause profound remodeling of the surrounding membrane. Specifically, scramblase activation causes the bilayer to thin at the open lipid pathway and the outer leaflet to bend towards the center of the bilayer at the dimer interface. We propose a model in which trans-bilayer lipid movement is enabled by these membrane and protein rearrangements and provide a general mechanistic framework for phospholipid scrambling.

Endoplasmic reticulum stress: major player in size-dependent inhibition of P-glycoprotein by silver nanoparticles in multidrug-resistant breast cancer cells

BackgroundDevelopment of multidrug resistance (MDR) is a major burden of successful chemotherapy, therefore, novel approaches to defeat MDR are imperative. Although the remarkable anti-cancer propensity of silver nanoparticles (AgNP) has been demonstrated and their potential application in MDR cancer has been proposed, the nanoparticle size-dependent cellular events directing P-glycoprotein (Pgp) expression and activity in MDR cancer have never been addressed. Hence, in the present study we examined AgNP size-dependent cellular features in multidrug resistant breast cancer cells.ResultsIn this study we report that 75 nm AgNPs inhibited significantly Pgp efflux activity in drug-resistant breast cancer cells and potentiated the apoptotic effect of doxorubicin, which features were not observed upon 5 nm AgNP treatment. Although both sized AgNPs induced significant ROS production and mitochondrial damage, 5 nm AgNPs were more potent than 75 nm AgNPs in this respect, therefore, these effects can not to be accounted for the reduced transport activity of ATP-driven pumps observed after 75 nm AgNP treatments. Instead we found that 75 nm AgNPs depleted endoplasmic reticulum (ER) calcium stores, caused notable ER stress and decreased plasma membrane positioning of Pgp.ConclusionOur study suggests that AgNPs are potent inhibitors of Pgp function and are promising agents for sensitizing multidrug resistant breast cancers to anticancer drugs. This potency is determined by their size, since 75 nm AgNPs are more efficient than smaller counterparts. This is a highly relevant finding as it renders AgNPs attractive candidates in rational design of therapeutically useful agents for tumor targeting. In the present study we provide evidence that exploitation of ER stress can be a propitious target in defeating multidrug resistance in cancers.

ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1

The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellularsubstrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.

Inhibition of blood-brain barrier efflux transporters promotes seizure in pregnant rats: Role of circulating factors

Seizure-provoking factors circulate late in gestation during normal pregnancy, but do not readily gain access to the brain due to the protective nature of the blood-brain barrier. In particular, efflux transporters are powerful ATP-driven pumps that actively prevent unwanted compounds from entering the brain. We hypothesized that acute inhibition of efflux transporters at the blood-brain barrier would result in spontaneous seizures in pregnant rats. We further hypothesized that the blood-brain barrier protects the maternal brain from seizure by increasing expression and/or activity of p-glycoprotein (P-gp), a major efflux transporter. Main blood-brain barrier efflux transporters were inhibited in-vivo in nonpregnant (Nonpreg) and pregnant (Preg; d19) Sprague Dawley rats (n=8/group). Seizures were monitored in conscious animals for 8h via chronically implanted electroencephalography (EEG) electrodes in the hippocampus and motor cortex and time-synced video. P-gp activity was measured via a calcein accumulation assay in freshly isolated cortical and hippocampal capillaries from Preg (d20) and Nonpreg rats (n=8–16/group), to assess regional susceptibility to transporter inhibition. P-gp expression, capillary density, and microglial activation as a measure of neuroinflammation were quantified using immunohistochemistry (n=4–6/group). Efflux transporter inhibition elicited hippocampal seizures within 1h in 100% of Preg rats that was not associated with neuroinflammation or elevated tumor necrosis factor alpha (TNFα) or vascular endothelial growth factor (VEGF), but negatively correlated with levels of estradiol. Hippocampal seizures were considerably less prevalent in Nonpreg rats. However, behavioral seizures in the motor cortex developed of similar severity in both groups of rats, demonstrating regional heterogeneity in response to efflux transporter inhibition. Basal P-gp activity was similar between groups, however, exposure to serum from Preg rats significantly decreased P-gp activity in the hippocampus, but not cortex, compared to serum from Nonpreg rats (0.29±0.1units/s in Preg vs. 0.06±0.02units/s in Nonpreg rats; p<0.05) that was not associated with elevated TNFα or VEGF. Thus, pregnancy differentially increased the susceptibility of the hippocampus to seizures in response to blood-brain barrier efflux transporter inhibition that may be due to the inhibitory effect of circulating factors in pregnancy on P-gp activity in the hippocampus.

Comparative Histological Study on the Effect of Stem Cells, and Gene Modified Stem Cells in Experimentally-Induced Diabetes Type 1 Cardiomyopathy

Background and Objectives: Sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA2a) is the ATP-driven pump thattranslocates Ca2+ from the cytoplasm to the lumen of the sarcoplasmic reticulum. 25% of children and adolescents withtype 1 diabetes will develop diastolic dysfunction that results in part from a reduction in the activity of SERCA2a. Thepresent study aimed at investigating and comparing the therapeutic effect of Adipose Mesenchymal Stem Cells (AMSCs)with SERCA2a gene modified AMSCs in diabetes type 1 induced cardiomyopathy of adult male albino rat.Materials and Methods: Twenty-nine adult male albino rats were divided into: Donor group of 2 rats used to obtainAMSCs. Group I (Control group): 6 rats not exposed to diabetes induction. Group II (Diabetic group): 7 rats injectedwith streptozotocin (STZ) 50mg/Kg once. Group III ( (AMSCs group): 7 rats injected with AMSCs following inductionof diabetes. Group IV (SERCA2a Modified AMSCs Group): 7 rats injected with SERCA2a modified AMSCs, followinginduction of diabetes. All rats were sacrificed 8 weeks from start of experiment.Results: Morphological changes, indicating inflammation and degeneration, were found in the cardiac muscle of diabeticrats and regressed remarkably by AMSCs and SERCA2a modified AMSCs. The regression of morphological changes wasconfirmed by histological, immunohistochemical, morphometric and serological studies.Conclusion: The therapeutic effect of SERCA2a modified AMSCs in diabetes type 1 cardiomyopathy was moreremarkable than that of AMSCs.

Phosphorylation/Dephosphorylation Assays.

The P-type ATPases form an autophosphorylated intermediate with ATP, and its isomeric transition and hydrolysis are obligatory events in the ATP-driven pump and thus for the energy coupling. The analyses of these reactions are therefore crucial for understanding the mechanism of the pump function and diseases caused by its defects. Here we describe the methods to analyze these processes in the transport cycle with a representative member of P-type ATPase family, SERCA1a, sarco(endo)plasmic reticulum Ca(2+)-ATPase.

Emergence of persistent Aspergillus terreus colonisation in a child with cystic fibrosis

Cystic Fibrosis (CF) is the most common inherited life shortening condition affecting Caucasians. CF is characterised by mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for an ATP-driven pump that transports sodium and chloride ions across epithelial surfaces [1]. CF is a multiple organ disease; however up to 95% of morbidity and mortality is due to pulmonary infection. The CF lung has impaired mucociliary clearance and a build-up of thick mucus which creates an ideal environment to facilitate microbial colonisation. Excessive neutrophil recruitment and enhanced inflammation ensue which causes airway epithelial cell damage, decline in lung function and eventual respiratory failure. Isolation of filamentous fungi, in particular Aspergillus spp. is common in respiratory secretions from CF patients [2]. Aspergillus terreus is the third most common filamentous fungus isolated from CF adult airway samples, being detected in 1.9 to 6.2% of CF patients [3,4]. In our clinic, 3 of 159 paediatric CF patients tested were A. terreus positive which is in line with the published literature on adults with CF (unpublished data). A. terreus has been reported to cause ABPA [5,6], infective endocarditis [6], pulmonary mycetoma [6] and invasive aspergillosis (IA) [7]. Until recently Aspergillus species identification was not thought to be therapeutically important; however different species within the genus can exhibit varying levels of antifungal drug resistance [8] and virulence in in vivo infection models [9]. Invasive disease caused by A. terreus can be as severe as IA caused by Apergillus fumigatus however A. terreus is inherently resistant to Amphotericin B [10]. Additionally IA caused by A. terreus is associated with long-term persistence of conidia and liver degeneration [11]. For these reasons A. terreus has the potential to cause complications post-transplant for people with CF. Here we present a case of a child with CF with a polymicrobial community in the airways among which A. terreus emerged and persisted as a dominant species.

Rhizobial strains isolated from nodules of Medicago marina in southwest Spain are abiotic-stress tolerant and symbiotically diverse

The isolation and characterisation of nitrogen-fixing root nodule bacteria from Medicago marina, a tolerant legume species, were studied in two areas from southwest Spain. A total of 30 out of 82 isolates with distinct ERIC-PCR fingerprints were analysed on the basis of molecular (PCR-RFLP of the 16S-23S rDNA intergenic spacer region (IGS) with two endonucleases, analysis of the 16S rDNA and symbiotic nodC gene sequences, plasmid profiles and SDS-PAGE of LPS, including the partial sequence of the housekeeping gene glnII and the symbiotic gene nodA of some representatives), physiological (utilisation of sole carbon sources, tolerance to antibiotics, NaCl, heavy metals, temperature and pH) and symbiotic parameters (efficacy on M. marina, M. minima, M. murex, M. orbicularis, M. polymorpha, M. sativa and M. truncatula). All the bacteria isolated from M. marina nodules belonged to Ensifer meliloti, except for one strain that belonged to E. medicae. To determine the nodulation range of M. marina, 10 different Ensifer species were tested for their ability to nodulate on this plant. E. kummerowiae CCBAU 71714 and the E. medicae control strain M19.1 were the only Ensifer species tested that developed nitrogen-fixing nodules on this plant. Most of the M. marina-nodulating strains showed tolerance to stress factors and all of them shared the presence of a gene similar to cadA, a gene that encodes for a PIB-type ATPase, which is a transporter belonging to the large superfamily of ATP-driven pumps involved in the transport of metals across cell membranes.

Neuronal calcium signaling: function and dysfunction

Calcium (Ca(2+)) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca(2+) signaling pathways to couple the Ca(2+) signal to their biochemical machinery. Ca(2+) influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca(2+) from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca(2+). Inside the cell, Ca(2+) is controlled by the buffering action of cytosolic Ca(2+)-binding proteins and by its uptake and release by mitochondria. The uptake of Ca(2+) in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca(2+) from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na(+)/Ca(2+) exchanger in the plasma membrane also participates in the control of neuronal Ca(2+). The impaired ability of neurons to maintain an adequate energy level may impact Ca(2+) signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca(2+) signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca(2+) signaling in the most important neurological disorders will then be considered.

Plant Cell Biology: The ABC of Monolignol Transport

Lignins are complex aromatic heteropolymers that reinforce the cell walls of terrestrial plants. A new study identifies an ATP-binding cassette ABC transporter that pumps a monolignol lignin precursor across the plasma membrane.

Carbonylation Contributes to SERCA2a Activity Loss and Diastolic Dysfunction in a Rat Model of Type 1 Diabetes

OBJECTIVEApproximately 25% of children and adolescents with type 1 diabetes will develop diastolic dysfunction. This defect, which is characterized by an increase in time to cardiac relaxation, results in part from a reduction in the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), the ATP-driven pump that translocates Ca2+ from the cytoplasm to the lumen of the sarcoplasmic reticulum. To date, mechanisms responsible for SERCA2a activity loss remain incompletely characterized.RESEARCH DESIGN AND METHODSThe streptozotocin (STZ)-induced murine model of type 1 diabetes, in combination with echocardiography, high-speed video detection, confocal microscopy, ATPase and Ca2+ uptake assays, Western blots, mass spectrometry, and site-directed mutagenesis, were used to assess whether modification by reactive carbonyl species (RCS) contributes to SERCA2a activity loss.RESULTSAfter 6–7 weeks of diabetes, cardiac and myocyte relaxation times were prolonged. Total ventricular SERCA2a protein remained unchanged, but its ability to hydrolyze ATP and transport Ca2+ was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of SERCA2a. Mutating affected residues to mimic physio-chemical changes induced on them by RCS reduced SERCA2a activity. Preincubating with the RCS, methylglyoxal (MGO) likewise reduced SERCA2a activity. Mutating an impacted residue to chemically inert glutamine did not alter SERCA2a activity, but it blunted MGO's effect. Treating STZ-induced diabetic animals with the RCS scavenger, pyridoxamine, blunted SERCA2a activity loss and minimized diastolic dysfunction.CONCLUSIONSThese data identify carbonylation as a novel mechanism that contributes to SERCA2a activity loss and diastolic dysfunction during type 1 diabetes.

Apical Recycling of the Gastric Parietal Cell H,K-ATPase

The gastric parietal cell was the first system where a regulated membrane recycling hypothesis was proposed as the principal means for moving molecular transporters between cellular compartments. Upon stimulation, massive membrane flow from an endosomal compartment of tubulovesicle membranes to the apical secretory surface places the ATP-driven pumps in position to secrete a solution of strong acid in collaboration with several other membrane transporters. This review focuses on the membrane recycling pathway and proteins that support the recruitment and redistribution of H,K-ATPase-rich membranes, including those involved in signal transduction, membrane targeting, docking, and fusing, in addition to the integral role of the actin cytoskeleton and its associated proteins in the process of membrane recycling. Although much of the evidence discussed here comes from parietal cell studies, other physiological transport systems, as well as less complex cellular and in vitro models, are examined and cited for generality of principle.

Restoring Blood-Brain Barrier P-Glycoprotein Reduces Brain Amyloid-β in a Mouse Model of Alzheimer's Disease

Reduced clearance of amyloid-beta (Abeta) from brain partly underlies increased Abeta brain accumulation in Alzheimer's disease (AD). The mechanistic basis for this pathology is unknown, but recent evidence suggests a neurovascular component in AD etiology. We show here that the ATP-driven pump, P-glycoprotein, specifically mediates efflux transport of Abeta from mouse brain capillaries into the vascular space, thus identifying a critical component of the Abeta brain efflux mechanism. We demonstrate in a transgenic mouse model of AD [human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576 strain] that brain capillary P-glycoprotein expression and transport activity are substantially reduced compared with wild-type control mice, suggesting a mechanism by which Abeta accumulates in the brain in AD. It is noteworthy that dosing 12-week-old, asymptomatic hAPP mice over 7 days with pregnenolone-16alpha-carbonitrile to activate the nuclear receptor pregnane X receptor restores P-glycoprotein expression and transport activity in brain capillaries and significantly reduces brain Abeta levels compared with untreated control mice. Thus, targeting intracellular signals that up-regulate blood-brain barrier P-glycoprotein in the early stages of AD has the potential to increase Abeta clearance from the brain and reduce Abeta brain accumulation. This mechanism suggests a new therapeutic strategy in AD.

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.

A P-type ATPase importer that discriminates between essential and toxic transition metals

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts.

Effects of both glucose and IP3 concentrations on action potentials in pancreatic β-cells

Considering the ATP-driven (SERCA) pump flux as function of glucose concentration and the calcium flux from the endoplasmic reticulum (ER) through the IP(3)R channel, the calcium-based phantom bursting model (PBM) of beta-cells (Bertram and Sherman in Bull Math Biol 66:1313, 2004) is theoretically extended to discuss the effects of glucose and inositol 1,4,5-trisphosphate (IP(3)) concentration on the membrane potential activities. When IP(3) concentration is fixed, it is found that there is a critical glucose concentration at which electrical bursting oscillations transfer into spiking, and the critical concentration of glucose is increased with the increasing of IP(3) concentration. To get the bursting oscillations in beta-cells, our theoretical results show that the stimulatory glucose concentration should be more than 10 mM, which is consistent with the normal physiological IP(3) level. When the stochastic opening and closing of IP(3)R channels are considered, it is shown that the membrane potential oscillation transfers from spiking to bursting with the channel number decreasing, and the average cytosolic free Ca(2+) concentration is increased with the increase of glucose concentration.

Estimation of cytoplasmic nitrate and its electrochemical potential in barley roots using 13NO3− and compartmental analysis

13NO3 was used to determine the intracellular compartmentation of NO3 in barley roots (Hordeum vulgare cv. Klondike), followed by a thermodynamic analysis of nitrate transport. Plants were grown in one-tenth Johnson's medium with 1 mol m3 NO3 (NO3-grown plants) or 1 mol m3 NH4NO3 (NH4NO3-grown plants). The cytoplasmic concentrations of NO3 in roots were only approx. 3-6 mol m3 (half-time for exchange approx. 21 s) in both NO3 and NH4NO3 plants. These pool sizes are consistent with published nitrate microelectrode data, but not with previous compartmental analyses. The electrochemical potential gradient for nitrate across the plasmalemma was +26 +/-1 kJ mol1 in both NO3- and NH4NO3-grown plants, indicating active uptake of nitrate. At an external pH of 6, the plasmalemma electrochemical potential for protons would be approx. -29 +/- 4 kJ mol1. If the cytoplasmic pH was 7.3 +/- 0.2, then 2H+/1NO3 cotransport, or a primary ATP-driven pump (2NO3/1ATP), are both thermodynamically possible. NO3 is also actively transported across the tonoplast (approx. +6 to 7 kJ mol1).