Non-radioactive Rb+ Efflux Assay for Screening KATP Channel Modulators.

ATP-sensitive potassium (KATP) channels function as metabolic sensors that link cell membrane excitability to the cellular energy status by controlling potassium ion (K+) flow across the cell membrane according to intracellular ATP and ADP concentrations. As such, KATP channels influence a broad spectrum of physiological processes, including insulin secretion and cardiovascular functions. KATP channels are hetero-octamers, consisting of four inward rectifier potassium channel subunits, Kir6.1 or Kir6.2, and four sulfonylurea receptors (SURs), SUR1, SUR2A, or SUR2B. Different Kir6 and SUR isoforms assemble into KATP channel subtypes with distinct tissue distributions and physiological functions. Mutations in the genes encoding KATP channel subunits underlie various human diseases. Targeted treatment for these diseases requires subtype-specific KATP channel modulators. Rubidium ions (Rb+) also pass through KATP channels, and Rb+ efflux assays can be used to assess KATP channel function and activity. Flame atomic absorption spectroscopy (Flame-AAS) combined with microsampling can measure Rb+ in small volume, which provides an efficient tool to screen for compounds that alter KATP channel activity in Rb+ efflux assays. In this chapter, we describe a detailed protocol for Rb+ efflux assays designed to identify new KATP channel modulators with potential therapeutic utilities.

Mycorrhiza-mediated potassium transport in Medicago truncatula can be evaluated by using rubidium as a proxy.

Arbuscular mycorrhizal (AM) fungi considerably improve plant nutrient acquisition, particularly phosphorus and nitrogen. Despite the physiological importance of potassium (K+) in plants, there is increasing interest in the mycorrhizal contribution to plant K+ nutrition. Yet, methods to track K+ transport are often costly and limiting evaluation opportunities. Rubidium (Rb+) is known to be transported through same pathways as K+. As such our research efforts attempt to evaluate if Rb+ could serve as a viable proxy for evaluating K+ transport in AM symbiosis. Therefore, we examined the transport of K+ in Medicago truncatula colonized by the AM fungus Rhizophagus irregularis isolate 09 having access to various concentrations of Rb+ in custom-made two-compartment systems. Plant biomass, fungal root colonization, and shoot nutrient concentrations were recorded under sufficient and limited K+ regimes. We report that AM plants displayed higher shoot Rb+ and K+ concentrations and a greater K+:Na+ ratio relative to non-colonized plants in both sufficient and limited K+ conditions. Consequently, our results indicate that Rb+ can be used as a proxy to assess the movement of K+ in AM symbiosis, and suggest the existence of a mycorrhizal uptake pathway for K+ nutrition in M. truncatula.

Bioremediation potential of hexavalent chromium-resistant Arthrobacter globiformis 151B: study of the uptake of cesium and other alkali ions.

Cesium (Cs+) enters environments largely because of global release into the environment from weapons testing and accidents such as Fukushima Daiichi and Chernobyl nuclear waste. Even at low concentrations, Cs+ is highly toxic to ecological receptors because of its physicochemical similarity to macronutrient potassium (K+). We investigated the uptake and accumulation of Cs+ by Arthrobacter globiformis strain 151B in reference to three similar alkali metal cations rubidium (Rb+), sodium (Na+), and potassium (K+). The impact of hexavalent chromium (Cr+6) as a co-contaminant was also evaluated. A. globiformis 151B accumulated Cs+ and Cr6+ in a time-dependent fashion. In contrast, the uptake and accumulation of Rb+ did not exhibit any trends. An exposure to Cs+, Rb+, and Cr+6 triggered a drastic increase in K+ and Na+ uptake by the bacterial cells. That was followed by the efflux of K+ and Na+, suggesting a Cs+ "substitution." Two-dimensional gel-electrophoresis of bacterial cell proteomes with the following mass-spectrometry of differentially expressed bands revealed that incubation of bacterial cells with Cs+ induced changes in the expression of proteins involved in the maintenance of cellular homeostasis and reactive oxygen species removal. The ability of A. globiformis 151B to mediate the uptake and accumulation of cesium and hexavalent chromium suggests that it possesses wide-range bioremediation potential.

Rubidium Chloride Increases Life Span Through an AMPK/FOXO-Dependent Pathway in Caenorhabditis elegans.

AMP-activated protein kinase (AMPK) is involved in life-span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. AMPK is currently considered a promising target for preventing age-related diseases. Rubidium is one of the trace elements in the human body. As early as the 1970s, rubidium chloride (RbCl) was reported to have neuroprotective effects. In this work, we report the antiaging effect of RbCl in Caenorhabditis elegans. Specifically, we reveal that (a) RbCl does increase the life span and enhance stress resistance in C. elegans without disturbing their fecundity. (b) RbCl induces superoxide dismutase expression, which is essential for its antiaging and antistress effect. (c) AAK-2 and DAF-16 are essential to the antiaging efficacy of RbCl, and RbCl can promote DAF-16 translocating into the nucleus, suggesting that RbCl delays aging by regulating the AMPK/FOXO pathway. RbCl can be a promising agent against aging-related diseases.

Surprising Condensation Reactions of the Azadithiolate Cofactor.

Azadithiolate, a cofactor found in all [FeFe]-hydrogenases, is shown to undergo acid-catalyzed rearrangement. Fe2 [(SCH2 )2 NH](CO)6 self-condenses to give Fe6 [(SCH2 )3 N]2 (CO)17 . The reaction, which is driven by loss of NH4+ , illustrates the exchange of the amine group. X-ray crystallography reveals that three Fe2 (SR)2 (CO)x butterfly subunits interconnected by the aminotrithiolate [N(CH2 S)3 ]3- . Mechanistic studies reveal that Fe2 [(SCH2 )2 NR](CO)6 participate in a range of amine exchange reactions, enabling new methodologies for modifying the adt cofactor. Ru2 [(SCH2 )2 NH](CO)6 also rearranges, but proceeds further to give derivatives with Ru-alkyl bonds Ru6 [(SCH2 )3 N][(SCH2 )2 NCH2 ]S(CO)17 and [Ru2 [(SCH2 )2 NCH2 ](CO)5 ]2 S.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Šresolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Exposure to a natural nitric oxide donor negatively affects the potential influx of rubidium in potassium-starved Arabidopsis plants.

Nitric oxide (NO) and potassium (K+) exert a profound influence on the acclimation of plants to multiple stress conditions. A recent report indicated that exogenous addition of an NO donor causes, under conditions of adequate K+ supply, a detrimental effect on K+ status. It remains unknown whether an exogenous NO source could negatively affect the potential capture of this element when plants are faced with a K+ shortage. In this work we offer evidence that, under conditions of K+-deprivation, the addition of the naturally occurring NO donor, S-nitrosoglutathione (GSNO), diminishes the potential inward transport of the K+-analogue rubidium (Rb+) from diluted Rb+ concentrations in Arabidopsis thaliana. Studies with the akt1-2 mutant, lacking the AKT1 inward-rectifier K+-channel involved in K+-uptake, unveiled that the effect of GSNO on Rb+-influx involves a non-AKT1 component. In addition, exposure to the NO-donor led to down-regulation of transcripts coding for the AtHAK5 K+-transporter, a major component of the K+-transport machinery in K+-deprived plants. Moreover, studies with the hak5 mutant showed that GSNO could either stimulate Rb+-uptake or does not lead to a significant effect on Rb+-uptake relative to -K+ and to -K+ in the presence of decayed GSNO, respectively, thus indicating that the presence of AtHAK5 is required for GSNO exerting an inhibitory effect.

Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ channel.

Two-pore domain K+ (K2P) channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via an SF-mediated gating mechanism, but we found here that only very strong nonphysiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mm). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.

Assessment of Silver-Nanoparticles-Induced Erythrocyte Cytotoxicity through Ion Transport Studies.

BACKGROUND/AIMS: Silver nanoparticles (AgNPs) are the most frequently used nanomaterials in industrial and biomedical applications. Their functionalization significantly impacts their properties and potential applications. Despite the need to produce, investigate and apply them, not much is known about the toxicity of silver nanoparticles to and their interaction with blood components, such as erythrocytes. Here, we report on the effect of two negatively charged AgNPs (Creighton, and Lee-Meisel) on ion transport in human red blood cells (HRBCs). METHODS: HRBCs were obtained from blood of adult donors, which was either expired, fresh or refrigerated for variable lengths of time, and from fresh or refrigerated cord blood. Rb+ and K+ ions were measured by atomic emission and absorption spectrophotometry, respectively. Erythrocyte hemoglobin optical density (Hbc OD), was determined at 527 nm to estimate RBC volume in the same tubes in which Rb+ and K+ were measured. Cellular Rb+ uptake and intracellular K+ concentrations, [K]i, were calculated in mmol/L of original cells (LOC) per time. Rubidium, a potassium ion (K+) congener used to measure K+ influx, [K]i, and Hbc ODs were determined in the presence and absence of several concentrations (0-150 µg mL-1) of spherical AgNPs of an average diameter of 10 nm, at different time points (0-60 min). RESULTS: Creighton AgNPs inhibited Rb+ influx and depleted the cells of K+ independently of the source and in a time and dose-dependent manner. In contrast, Lee-Meisel AgNPs caused ~ 50 % Rb+ influx inhibition and ~ 15 % K+ loss with larger interindividual variability than Creighton AgNPs. The loss of K+ from HRBCs was entirely accounted for by an increase in extracellular K+ concentration, [K]o. Enhanced dark field optical microscopy in conjunction with CytoViva® hyperspectral imaging helped visualize AgNPs internalized by HRBCs, thus pointing to a potential cause for their cytotoxic effects. CONCLUSION: These findings indicate that HRBC K+ homeostasis is an early and sensitive biomarker for AgNPs toxicity and is a function of their surface functionalization.

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb.

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.

K⁺ and Rb⁺ Affinities of the Na,K-ATPase α1 and α2 Isozymes: An Application of ICP-MS for Quantification of Na⁺ Pump Kinetics in Myofibers.

The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K⁺ and Rb⁺ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2-/-). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb⁺ and K⁺ with comparable apparent affinities; however; turnover rate is reduced when Rb⁺ is the transported ion; (2) The rate of Rb⁺ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K⁺ affinity of the α2 isozymes for K⁺ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes are 1.3 and 4 mM, respectively. Collectively, these results validate the single fiber model for studies of Na,K-ATPase transport and kinetic constants, and they imply the existence of mechanisms that dynamically limit pump activity during periods of active transport.

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.

Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations.

Exomer is an adaptor complex required for the direct transport of a selected number of cargoes from the trans-Golgi network (TGN) to the plasma membrane in Saccharomyces cerevisiae However, exomer mutants are highly sensitive to increased concentrations of alkali metal cations, a situation that remains unexplained by the lack of transport of any known cargoes. Here we identify several HAL genes that act as multicopy suppressors of this sensitivity and are connected to the reduced function of the sodium ATPase Ena1. Furthermore, we find that Ena1 is dependent on exomer function. Even though Ena1 can reach the plasma membrane independently of exomer, polarized delivery of Ena1 to the bud requires functional exomer. Moreover, exomer is required for full induction of Ena1 expression after cationic stress by facilitating the plasma membrane recruitment of the molecular machinery involved in Rim101 processing and activation of the RIM101 pathway in response to stress. Both the defective localization and the reduced levels of Ena1 contribute to the sensitivity of exomer mutants to alkali metal cations. Our work thus expands the spectrum of exomer-dependent proteins and provides a link to a more general role of exomer in TGN organization.

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel.

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1-S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+ These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity.

Origin of Ion Specificity of Telomeric DNA G-Quadruplexes Investigated by Free-Energy Simulations.

Telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) that form G-quadruplex structures made of stacked guanines with monovalent cations bound at a central cavity. Although different ions can stabilize a G-quadruplex structure, the preferred bound ions are typically K+ or Na+. Several different strand-folding topologies have been reported for Q-quadruplexes formed from telomeric repeats depending on DNA length and ion solution condition. This suggests a possible dependence of the ion selectivity of the central pore on the folding topology of the quadruplex. Molecular dynamics free energy perturbation has been employed to systematically study the relative affinity of the central quadruplex pore for different cation types and the associated energetic and solvation contributions to ion selectivity. The calculations have been performed on two different common quadruplex folding topologies. For both topologies, the same ion selectivity was found with a preference for K+ followed by Rb+ and Na+, which agrees with the experimentally determined preference for most investigated quadruplexes. The selectivity is determined by a balance between attractive Coulomb interactions and loss of hydration but also modulated by van der Waals contributions. Specificity is mediated by the central guanines and no significant contribution of the nucleic acid backbone. The simulations indicate that different topologies might be stabilized by ions bound at the surface or alternative sites of the quadruplex because the ion specificity of the central pore does not depend on the strand folding topology.

Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium.

Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2-fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates pI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. J. Cell. Physiol. 232: 1489-1500, 2017. © 2016 Wiley Periodicals, Inc.

Rubidium and potassium levels are altered in Alzheimer's disease brain and blood but not in cerebrospinal fluid.

Loss of intracellular compartmentalization of potassium is a biochemical feature of Alzheimer's disease indicating a loss of membrane integrity and mitochondrial dysfunction. We examined potassium and rubidium (a biological proxy for potassium) in brain tissue, blood fractions and cerebrospinal fluid from Alzheimer's disease and healthy control subjects to investigate the diagnostic potential of these two metal ions. We found that both potassium and rubidium levels were significantly decreased across all intracellular compartments in the Alzheimer's disease brain. Serum from over 1000 participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), showed minor changes according to disease state. Potassium and rubidium levels in erythrocytes and cerebrospinal fluid were not significantly different according to disease state, and rubidium was slightly decreased in Alzheimer's disease patients compared to healthy controls. Our data provides evidence that contrasts the hypothesized disruption of the blood-brain barrier in Alzheimer's disease, with the systemic decrease in cortical potassium and rubidium levels suggesting influx of ions from the blood is minimal and that the observed changes are more likely indicative of an internal energy crisis within the brain. These findings may be the basis for potential diagnostic imaging studies using radioactive potassium and rubidium tracers.

Labeling Feral Spruce Budworm (Lepidoptera: Tortricidae) Populations With Rubidium.

Rubidium (Rb) is a trace element that occurs naturally in low concentrations and is easily absorbed by plants, making it a useful tool for labeling insect defoliators, such as spruce budworm (Choristoneura fumiferana Clemens). Balsam fir trees (Abies balsamea (L.) Miller) injected with either 8 or 16 g per tree of rubidium chloride (RbCl) showed quick uptake and distribution throughout the crown, with no negative effects on tree shoot growth or spruce budworm survival and development. Adult spruce budworm that fed as larvae on trees injected with RbCl were clearly labeled, with significantly higher Rb concentrations than the background levels found in adults that fed as larvae on control trees. Rb concentrations in feral spruce budworm adults for both the 8 g (9 µg/g) and 16 g (25 µg/g) per tree treatments were at least five times lower than those in laboratory-reared adults on 1,000 µg/g RbCl diet (125 µg/g); survival, development, pupal weight, sex ratio, and mating status of spruce budworm were not adversely affected by Rb treatment. Egg masses laid by feral females that fed as larvae on Rb-labeled trees were also labeled with Rb. Injecting trees with RbCl is a viable technique for labeling feral spruce budworm populations to help distinguish local populations from immigrants to better evaluate the success of early intervention strategies such as mating disruption.

The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

Marking Drosophila suzukii (Diptera: Drosophilidae) With Rubidium or 15N.

Drosophila suzukii Matsumura (Diptera: Drosophilidae) has caused significant economic damage to berry and stone fruit production regions. Markers that are systemic in plants and easily transferred to target organisms are needed to track D. suzukii exploitation of host resources and trophic interactions. High and low concentrations of the trace element, rubidium (Rb), and the stable isotope, 15N, were tested to mark D. suzukii larvae feeding on fruits of enriched strawberry plants grown in containers under greenhouse conditions. Fly marker content and proportion of flies marked 1, 7, and 14 d after emergence from enriched fruits and fly dry mass were analyzed. Nearly 100% of the flies analyzed 14 d after emerging from 15N-enriched plants were marked, whereas only 30-75% and 0-3% were marked 14 d after emerging from high and low Rb concentration plants, respectively. Rapid Rb decay, strong 15N persistence, and the economics of using these markers in the field to elucidate D. suzukii pest ecology are discussed.