Increase In Transport Activity Research Articles

Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition

The potassium chloride cotransporter KCC2 is crucial for Cl- extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr934Ala/Ser937Asp double mutation, in which Ser937 is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr934 arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl−]i. and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr934Ala/Ser937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr934Ala/Ser937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized ECl−. These data demonstrate that phospho-regulation of KCC2 Ser937 is a potent way to interfere with the excitation-inhibition balance in neural circuits.

Distinct ClC-6 and ClC-7 Cl- sensitivities provide insight into ClC-7's role in lysosomal Cl- homeostasis.

ClC-6 and ClC-7 are closely related, intracellular Cl- /H+ antiporters belonging to the CLC family of channels and transporters. They localize to acidic late endosomes and lysosomes and probably function in ionic homeostasis of these contiguous compartments. ClC-7 transport function requires association with the accessory protein Ostm1, whereas ClC-6 transport does not. To elucidate their roles in endo-lysosomes, we measured Cl- - and pH-dependences of over-expressed wild-type ClC-6 and ClC-7, as well as disease-associated mutants, using high-resolution recording protocols. Lowering extracellular Cl- (corresponding to luminal Cl- in endo-lysosomes) reduced ClC-6 currents, whereas it increased transport activity of ClC-7/Ostm1. Low extracellular Cl- activated ClC-7/Ostm 1 under acidic extracellular conditions, as well as under conditions of low intracellular chloride. Activation is conserved in ClC-7Y713C , a variant displaying disrupted PI(3,5)P2 inhibition. Detailed biophysical analysis of disease-associated ClC-6 and ClC-7 gain-of-function (GoF) variants, ClC-6Y553C and ClC-7Y713C , and the ClC-7Y577C and ClC-6Y781C correlates, identified additional functional nuances distinguishing ClC-6 and ClC-7. ClC-7Y577C recapitulated GoF produced by ClC-6Y553C . ClC-6Y781C displayed transport activation qualitatively similar to ClC-7Y713C , although current density did not differ from that of wild-type ClC-6. Finally, rClC-7R760Q , homologous to hClC-7R762Q , an osteopetrosis variant with fast gating kinetics, appeared indifferent to extracellular Cl- , identifying altered Cl- sensitivity as a plausible mechanism underlying disease. Collectively, the present studies underscore the distinct roles of ClC-6 and ClC-7 within the context of their respective localization to late endosomes and lysosomes. In particular, we suggest the atypical inhibition of ClC-7 by luminal Cl- serves to limit excessive intraluminal Cl- accumulation. KEY POINTS: ClC-6 and ClC-7 are late endosomal and lysosomal 2 Cl- /1 H+ exchangers, respectively. When targeted to the plasma membrane, both activate slowly at positive voltages. ClC-6 activity is decreased in low extracellular (i.e. luminal) chloride, whereas ClC-7 is activated by low luminal chloride, even at acidic pH. The functional gain-of-function phenotypes of the ClC-6 and ClC-7 disease mutations ClC-6Y553C and ClC-7Y715C are maintained when introduced in their respective homologues, ClC-7Y577C and ClC-6Y781C , with all mutations retaining chloride dependence of the respective wild type (WT). An osteopetrosis mutation of ClC-7 displaying fast gating kinetics (R762Q) was less sensitive to extracellular chloride compared to WT. The opposing substrate dependences of ClC-6 and ClC-7 Cl- / H+ exchangers point to non-overlapping physiological functions, leading us to propose that inhibition of ClC-7 by luminal chloride and protons serves to prevent osmotic stress imposed by hyper-accumulation of chloride.

Changes in adolescent physical activity levels pre and post COVID-19 in Ireland

Abstract Background The World Health Organisation (WHO) recommends moderate to vigorous physical activity (MVPA) for at least, on average, 60 minutes per day, and muscle strengthening exercises at least three times a week for adolescent health. Lockdowns, sports club and school closures during COVID-19 limited children's PA opportunities. The aim of this study was to examine changes in PA behaviour among students in schools undertaking a PA intervention (the Active School Flag: ASF) over three consecutive years. Methods ASF is a whole school intervention. Its aim is to increase student PA opportunities and thus, PA levels. ASF implementation team includes students, teachers and school management. Using a repeated cross-sectional panel study design, all students in five pilot ASF schools were invited to complete the ASF whole-school survey in September 2019, 2020 and 2021. Descriptive statistics, contingency tables and multi-logistic regression analyses evaluated changes in PA behaviour over time. Results Students in five schools (N = 7530, 33% male; 65% female, 2% other) took part. Physical inactivity (WHO guidelines for only 0-2days) dropped from 25% in 2019 to 22% in 2020 and 23% in 2021. Analysis by gender showed females to be most active in 2020 (p = .004). No significant changes, over time, were found for muscle-strengthening exercises. Four out of the five schools reported a significant increase in active travel and a decrease in car transport behaviour from 2019-2021, no gender differences were found. Conclusions COVID-19 public health restrictions varied over the duration of this study (higher in 2020 than at other time points). Little or no change in overall PA or muscle-strengthening exercises was established, yet significant increases in active transport to and from school were reported. Further research should explore mechanisms and the potential of a displacement hypothesis for types of PA change during this time. Key messages • COVID-19 movement restrictions, such as lockdowns, school and sports club closures contributed to increases in active transport to and from school, while other forms of PA remained largely unchanged. • The Active School Flag may be an effective programme to reduce the amount of students who are physically inactive.

Nocturnal increase in cerebrospinal fluid secretion as a circadian regulator of intracranial pressure

BackgroundIt is crucial to maintain the intracranial pressure (ICP) within the physiological range to ensure proper brain function. The ICP may fluctuate during the light-dark phase cycle, complicating diagnosis and treatment choice in patients with pressure-related disorders. Such ICP fluctuations may originate in circadian or sleep-wake cycle-mediated modulation of cerebrospinal fluid (CSF) flow dynamics, which in addition could support diurnal regulation of brain waste clearance.MethodsICP was monitored continuously in patients who underwent placement of an external ventricular drain (EVD) and by telemetric monitoring in experimental rats. CSF was collected via the EVD in patients and the rodent CSF secretion rate determined by in vivo experimentation. Rodent choroid plexus transporter transcripts were quantified with RNAseq and transport activity with ex vivo isotope transport assays.ResultsWe demonstrated that ICP increases by 30% in the dark phase in both species, independently of vascular parameters. This increase aligns with elevated CSF collection in patients (12%) and CSF production rate in rats (20%), the latter obtained with the ventriculo-cisternal perfusion assay. The dark-phase increase in CSF secretion in rats was, in part, assigned to increased transport activity of the choroid plexus Na+,K+,2Cl- cotransporter (NKCC1), which is implicated in CSF secretion by this tissue.ConclusionCSF secretion, and thus ICP, increases in the dark phase in humans and rats, irrespective of their diurnal/nocturnal activity preference, in part due to altered choroid plexus transport activity in the rat. Our findings suggest that CSF dynamics are modulated by the circadian rhythm, rather than merely sleep itself.

The Ubiquitin E3 Ligase Nedd4 Regulates the Expression and Amyloid-β Peptide Export Activity of P-Glycoprotein.

The ATP-binding cassette transporter, P-glycoprotein (P-gp), has been demonstrated to facilitate the clearance of amyloid-beta (Aβ) peptides, exporting the neurotoxic entity out of neurons and out of the brain via the blood–brain barrier. However, its expression and function diminish with age and in Alzheimer’s disease. P-gp is known to undergo ubiquitination, a post-translational modification that results in internalisation and/or degradation of the protein. NEDD4-1 is a ubiquitin E3 ligase that has previously been shown to ubiquitinate P-gp and reduce its cell surface expression. However, whether this effect translates into altered P-gp activity remains to be determined. siRNA was used to knockdown the expression of Nedd4 in CHO-APP cells. Western blot analysis confirmed that absence of Nedd4 was associated with increased P-gp protein expression. This was accompanied by increased transport activity, as shown by export of the P-gp substrate calcein-AM, as well as enhanced secretion of Aβ peptides, as shown by ELISA. These results implicate Nedd4 in the regulation of P-gp, and highlight a potential approach for restoring or augmenting P-gp expression and function to facilitate Aβ clearance from the brain.

An endoplasmic reticulum–localized cytochrome b5 regulates high-affinity K+ transport in response to salt stress in rice

Potassium (K+) is an essential element for growth and development in both animals and plants, while high levels of environmental sodium (Na+) represent a threat to most plants. The uptake of K+ from high-saline environments is an essential mechanism to maintain intracellular K+/Na+ homeostasis, which can help reduce toxicity caused by Na+ accumulation, thereby improving the salt tolerance of plants. However, the mechanisms and regulation of K+-uptake during salt stress remain poorly understood. In this study, we identified an endoplasmic reticulum-localized cytochrome b5 (OsCYB5-2) that interacted with a high-affinity K+ transporter (OsHAK21) at the plasma membrane. The association of OsCYB5-2 with the OsHAK21 transporter caused an increase in transporter activity by enhancing the apparent affinity for K+-binding but not Na+-binding. Heme binding to OsCYB5-2 was essential for the regulation of OsHAK21. High salinity directly triggered the OsHAK21-OsCYB5-2 interaction, promoting OsHAK21-mediated K+-uptake and restricting Na+ entry into cells; this maintained intracellular K+/Na+ homeostasis in rice cells. Finally, overexpression of OsCYB5-2 increased OsHAK21-mediated K+ transport and improved salt tolerance in rice seedlings. This study revealed a posttranslational regulatory mechanism for HAK transporter activity mediated by a cytochrome b5 and highlighted the coordinated action of two proteins to perceive Na+ in response to salt stress.

Pnictogen-Bonding Catalysis and Transport Combined: Polyether Transporters Made In Situ

The combination of catalysis and transport across lipid bilayer membranes promises directional access to a solvent-free and structured nanospace that could accelerate, modulate, and, at best, enable new chemical reactions. To elaborate on these expectations, anion transport and catalysis with pnictogen and tetrel bonds are combined with polyether cascade cyclizations into bioinspired cation transporters. Characterized separately, synergistic anion and cation transporters of very high activity are identified. Combined for catalysis in membranes, cascade cyclizations are found to occur with a formal rate enhancement beyond one million compared to bulk solution and product formation is detected in situ as an increase in transport activity. With this operational system in place, intriguing perspectives open up to exploit all aspects of this unique nanospace for important chemical transformations.

Investigation of the Effect of the Deck Material on the Cost in Cable-Stayed Bridges with Different Spans

Abstract Recent developments in the social sphere also cause an increase in transportation activities. Increased transport activities lead to the construction of new roads and bridges. Different bridge construction systems are available to overcome large span obstacles. Cable-stayed bridges are more advantageous construction systems than other bridge type building carrier systems in overcoming large spans through suspension cables. Therefore, it is also widely preferred by designers. The biggest factor in the development of cable-stayed bridges is undoubtedly steel cables. Cable-stayed bridges are bridge structures that become lighter with the increase of the span, which has a more expanded flexibility, and that includes a cable system with the effect of nonlinear factors. Costs of cable-stayed bridges vary according to different spans. The span as well as the deck material used in the bridge system have a great effect on the cost. In cable-stayed bridge systems, decks are constructed of reinforced concrete and steel. The costs of cable-stayed bridges are widely discussed around the world; therefore, the effect of the span and deck material on the cost of cable-stayed bridges is being investigated. The main bearing elements of such bridges are cables, decks, and towers, and among these elements, the tower bridge carries all the weight of the bridge, even other external loads such as vehicle, wind, etc. In this study, the three-dimensional model of the cable, deck and tower elements that make up the cable-stayed bridge system was created and analysed using the CSI Bridge Program. The AASHTO LRFD Standards, which are widely used in the analysis of bridge systems with the CSI Bridge program and the design of bridge systems in the world, were used. In the study, the analysis and designs of cable-stayed bridges with reinforced concrete and steel deck at 250, 500, 750, 1000, 1500, 2000 meters span were carried out. The amount of materials and costs used in the analysis and design of the cable-stayed bridge systems were obtained and the results were interpreted.

New insights into the acetate uptake transporter (AceTr) family: Unveiling amino acid residues critical for specificity and activity

Aiming at improving the transport of biotechnologically relevant carboxylic acids in engineered microbial cell factories, the focus of this work was to study plasma membrane transporters belonging to the Acetate Uptake Transporter (AceTr) family. Ato1 and SatP, members of this family from Saccharomyces cerevisiae and Escherichia coli, respectively, are the main acetate transporters in these species. The analysis of conserved amino acid residues within AceTr family members combined with the study of Ato1 3D model based on SatP, was the rationale for selection of site-directed mutagenesis targets. The library of Ato1-GFP mutant alleles was functionally analysed in the S. cerevisiae IMX1000 strain which shows residual growth in all carboxylic acids tested. A gain of function phenotype was found for mutations in the residues F98 and L219 located at the central constrictive site of the pore, enabling cells to grow on lactic and on succinic acid. This phenotype was associated with an increased transport activity for these substrates. A dominant negative acetic acid hypersensitivity was induced in S. cerevisiae cells expressing the E144A mutant, which was associated with an increased acetic acid uptake. By utilizing computer-assisted 3D-modelling tools we highlight structural features that explain the acquired traits found in the analysed Ato1 mutants. Additionally, we achieved the proper expression of the Escherichia coli SatP, a homologue of Ato1, in S. cerevisiae. To our knowledge, this constitutes the first report of a fully functional bacterial plasma membrane transporter protein in yeast cells.

1,25(OH)2 vitamin D3 stimulates active phosphate transport but not paracellular phosphate absorption in mouse intestine.

Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi-IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi-IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi-IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH)2 vitamin D3 to wild-type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal-depleted Slc34a2-deficient mice. In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH. While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. Intestinal absorption of phosphate is stimulated by 1,25(OH)2 vitamin D3. At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na+ /phosphate cotransporter NaPi-IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)2 vitamin D3 stimulates NaPi-IIb expression and function, and loss of NaPi-IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi-IIb is the only target for hormonal regulation by 1,25(OH)2 vitamin D3 . Here we compared the effects of intraperitoneal administration of 1,25(OH)2 vitamin D3 (2days, once per day) in wild-type and intestinal-specific Slc34a2-deficient mice, and analysed trans- vs. paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild-type mice, though NaPi-IIb protein expression was upregulated in jejunum and ileum. In contrast, 1,25(OH)2 vitamin D3 administration had no effect in Slc34a2-deficient mice, suggesting that the hormone specifically regulates NaPi-IIb expression. In both groups, 1,25(OH)2 vitamin D3 elicited the expected increase of plasma fibroblast growth factor 23 (FGF23) and reduction of parathyroid hormone (PTH). Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphataemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only higher FGF23 can explain the renal response in Slc34a2-deficient mice. Thus, 1,25(OH)2 vitamin D3 stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi-IIb while the paracellular pathway appears not to be affected.

Carbon export from leaves is controlled via ubiquitination and phosphorylation of sucrose transporter SUC2

All multicellular organisms keep a balance between sink and source activities by controlling nutrient transport at strategic positions. In most plants, photosynthetically produced sucrose is the predominant carbon and energy source, whose transport from leaves to carbon sink organs depends on sucrose transporters. In the model plant Arabidopsis thaliana, transport of sucrose into the phloem vascular tissue by SUCROSE TRANSPORTER 2 (SUC2) sets the rate of carbon export from source leaves, just like the SUC2 homologs of most crop plants. Despite their importance, little is known about the proteins that regulate these sucrose transporters. Here, identification and characterization of SUC2-interaction partners revealed that SUC2 activity is regulated via its protein turnover rate and phosphorylation state. UBIQUITIN-CONJUGATING ENZYME 34 (UBC34) was found to trigger turnover of SUC2 in a light-dependent manner. The E2 enzyme UBC34 could ubiquitinate SUC2 in vitro, a function generally associated with E3 ubiquitin ligases. ubc34 mutants showed increased phloem loading, as well as increased biomass and yield. In contrast, mutants of another SUC2-interaction partner, WALL-ASSOCIATED KINASE LIKE 8 (WAKL8), showed decreased phloem loading and growth. An in vivo assay based on a fluorescent sucrose analog confirmed that SUC2 phosphorylation by WAKL8 can increase transport activity. Both proteins are required for the up-regulation of phloem loading in response to increased light intensity. The molecular mechanism of SUC2 regulation elucidated here provides promising targets for the biotechnological enhancement of source strength.

Intermittent Aeolian Saltation Over a Gobi Surface: Threshold, Saltation Layer Height, and High‐Frequency Variability

AbstractGobi is a type of desert pavement in Asia over which aeolian saltation generates atmospheric dust. However, the high‐frequency behavior of aeolian saltation over gobi is still poorly understood. In this study, coupled high‐frequency observations of three‐dimensional (3‐D) wind speed (U) and saltation particle count rate in a vertical array of Sensit sensors were performed in a gobi region of Milan County, southern Xinjiang, China. The results revealed that aeolian saltation over a gobi surface exhibited an intermittent phenomenon, and the level of the intermittency was governed by the relative wind strength (s), which is consistent with that of sand surfaces. The threshold wind speed (Ut) decreased exponentially with increasing transport activity parameter (AP), and a prominent decrease in Ut was examined when the AP values reached 0.7; comparatively, for sand surfaces, a linearly declining trend occurred over the whole AP range of 0–1. The saltating particles over the gobi surface were distributed in layers of 0.08–0.30 m with an average value of 0.20 ± 0.04 m, which was significantly larger than that of sand surfaces. The relationship between the instantaneous sand transport rate (Q) and U over the gobi surface was also relatively poor and could be improved by lagging Q by 2 s. The mechanism for the collision between saltating particles and the gobi bed, which is rigid and complex in texture, was significantly different from that of the soft and deforming sand bed, causing the difference in aeolian saltation between gobi and sand surfaces.

Aryl hydrocarbon receptor ligands increase ABC transporter activity and protein expression in killifish (Fundulus heteroclitus) renal proximal tubules.

Many widespread and persistent organic pollutants, for example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and some polychlorinated biphenyls, activate the aryl hydrocarbon receptor (AhR) causing it to translocate to the cell nucleus where it transactivates target genes, increasing expression of a number of xenobiotic metabolizing enzymes as well as some transporters. AhR's ability to target transporters within the kidney is essentially unexplored. We show here that exposing isolated killifish (Fundulus heteroclitus) renal proximal tubules to micromolar β-naphthoflavone (BNF) or nanomolar TCDD roughly doubled the transport activity of Multidrug resistance-associated proteins Mrp2 and Mrp4, P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp), all ATP-driven xenobiotic efflux pumps and critical determinants of renal xenobiotic excretion. These effects were abolished by actinomycin D and cycloheximide and by the AhR antagonist, α-naphthoflavone, indicating that increased transport activity was dependent on transcription and translation as well as ligand binding to AhR. Quantitative immunostaining of renal tubules exposed to BNF and TCDD showed increased luminal membrane expression of Mrp2, Mrp4, P-gp and Bcrp. Thus, in these renal tubules, the four ABC transporters are targets of AhR action.

Tetrabromobisphenol A (TBBPA) Alters ABC Transport at the Blood-Brain Barrier.

Tetrabromobisphenol A (TBBPA, CAS No. 79-94-7) is a brominated flame retardant used in 90% of epoxy coated circuit boards. Exposures to TBBPA can induce neurotoxicity and disrupt MAPK, estrogen, thyroid, and PPAR-associated signaling pathways. Because these pathways also regulate transporters of the central nervous system barriers, we sought to determine the effect of TBBPA on the expression and activity of 3 ATP binding cassette (ABC) transporters of the blood-brain barrier (BBB). Using a confocal based assay, we measured the ex vivo and in vivo effects of TBBPA on P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) transport activity in rat brain capillaries. Our rationale for using a rat model was based on tissue availability, ease of handling, and availability of historical TBBPA toxicokinetic data. We found that TBBPA (1-1000 nM) exposure had no significant effect on multidrug resistance-associated protein 2 transport activity in either sex, suggesting TBBPA does not compromise the physical integrity of the BBB. However, low concentrations of TBBPA (1-100 nM) significantly decreased breast cancer resistant protein transport activity in both sexes. Additionally, TBBPA exposures (1-100 nM), elicited a sex-dependent response in P-gp transport: increasing transport activity in males and decreasing transport activity in females. All TBBPA dependent changes in transport activity were dose- and time-dependent. Inhibitors of either transcription or translation abolished the TBBPA dependent increases in male P-gp transport activity. Western blot and immunofluorescent assays confirmed the TBBPA dependent P-gp increases expression in males and decreases in females. Antagonizing PPAR-γ abolished the TBBPA dependent increases in males but not the decreases in females. However, the decreases in female P-gp transport were blocked by an ER-α antagonist. This work indicates that environmentally relevant concentrations of TBBPA (1-100 nM) alter ABC transporter function at the BBB. Moreover, permeability changes in the BBB can alter brain homeostasis, hinder central nervous system drug delivery, and increase the brain's exposure to harmful xenobiotic toxicants.

Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

The pivotal role of K+-Cl- cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

Isoform-Selective Targeting of Facilitative Glucose Transporters with Small Molecule Inhibitors

There are currently 14 known mammalian facilitative glucose transporters (GLUTs) that serve a variety of critical roles in maintaining glucose homeostasis. The ability to potently target glucose transport in an isoform-selective manner is of paramount importance to investigate the normal function of these transporter isoforms and their individual contributions to disease pathogenesis. A number of GLUT isoform-selective small molecule inhibitors have been identified. To investigate the structural basis of GLUT inhibition and facilitate further refinement of the efficacy and selectivity of these antagonists, we performed kinetic analysis of the effects of WBZ-117, C-20, BAY-876 and several HIV protease inhibitors, on 3H-2-deoxyglucose uptake into cell lines selectively over-expressing GLUT1, GLUT4 or a T30I H160N GLUT1 mutant transporter. Several important findings were made: 1) based on Ki(app) measurements, indinavir, lopinavir, WZB-117 and C-20 selectively inhibit GLUT4 13-55 fold vs. GLUT1. Although structurally divergent, the potency of these 4 compounds against GLUT1 can be markedly enhanced to levels approximating those for GLUT4 by substituting just two GLUT1 amino acids located in the glucose permeation pathway to those found in GLUT4 (T30I and H160N); 2) nelfinavir is a weak GLUT4 inhibitor but acts as a GLUT1 activator, increasing transport activity 1.4-fold; 3) saquinavir selectively inhibits GLUT4 and the T30I H160N GLUT1 mutant, and exhibits complex kinetic patterns in the mutant cell line consistent with a two site model for partial inhibition; 4) kinetic analysis revealed that selective GLUT1 inhibitor, BAY-876, is a noncompetitive inhibitor of this transporter. Taken together, these data provide key structural insights into the ability of small molecules to alter GLUT function that will aid in the development of novel isoform-selective drugs for glucose-dependent diseases. Disclosure M. Heitmeier: None. R.C. Hresko: None. M. Shanmugam: None. P.W. Hruz: None.

Obesity‐related health impacts of active transport policies in Australia – a policy review and health impact modelling study

ObjectiveTo review Australian policies on active transport, defined as walking and cycling for utilitarian purposes. To estimate the potential health impact of achieving four active transport policy scenarios. MethodsA policy review was undertaken, using key words to search government websites. Potential health benefits were quantified using a cohort simulation Markov model to estimate obesity and transport injury‐related health effects of an increase in active transport. Health adjusted life years (HALYs) gained and healthcare cost savings from diseases averted were estimated. Budget thresholds to achieve cost‐effectiveness were estimated for each scenario. ResultsThere is broad recognition of the health‐related benefits of active transport from all levels of Australian government. Modelling results suggest significant health‐related benefits of achieving increased prevalence of active transport. Total HALYs saved assuming a one‐year effect ranged from 565 (95%UI 173–985) to 12,105 (95%UI 4,970–19,707), with total healthcare costs averted ranging from $6.6M (95%UI $1.9M‐11.3M) to $141.2M (95%UI $53.8M–227.8M). ConclusionEffective interventions that improve rates of active transport may result in substantial healthcare‐related cost savings through a decrease in conditions related to obesity. Implications for public healthSignificant potential exists for effective and cost‐effective interventions that result in more walking and cycling.

Evaluation of Neonatal Transport in Western Switzerland: A Model of Perinatal Regionalization.

Neonatal transport is an essential part of regionalization for highly specialized neonatal intensive care. This retrospective analysis of prospectively collected data on neonatal transport activity in a large Swiss perinatal network more than 1 year, aimed to quantify this activity, to identify the needs for staff, and the demands regarding know-how and equipment. Of the 565 admissions to the tertiary neonatology clinic, 176 (31.2%) were outborn patients, transported as emergencies to the level III unit. In 71.6% of cases, respiratory insufficiency was one of the reasons for transfer. Circadian and weekly distribution showed increased transport activity on workdays between 8 am and 10 pm, but regular demands for emergency transports regardless of the time frame require a neonatal transport team available 24/7. This study highlights the importance of neonatal transport and unveils several functional and infrastructural insufficiencies, which led to suggestions for improvement.

Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.

Protein phosphatase PP2A - a novel interacting partner of carnitine transporter OCTN2 (SLC22A5) in rat astrocytes.

l-Carnitine is essential for translocation of fatty acids for their mitochondrial β-oxidation, a process shown in the brain to take place in astrocytes. Organic cation and carnitine plasma membrane transporter OCTN2 (SLC22A5) is present in astrocytes. OCTN2 activity and localization were previously shown to be regulated by protein kinase C (PKC), although no phosphorylation of the transporter was detected. In this study, mass spectrometry was used to identify rOctn2-interacting partners in astrocytes: several cytoskeletal, ribosomal, mitochondrial, heat-shock proteins, as well as proteins involved in trafficking and signaling pathways. The analysis of signaling proteins shows that Octn2 co-precipitated with PP2A phosphatase catalytical (C) and structural (A) subunits, and with its regulatory B"' subunits - striatin, SG2NA, and zinedin. The Octn2/PP2A complex is mainly detected in endoplasmic reticulum. PKC activation increases both, carnitine transport and, as shown by immunofluorescence and surface biotinylation, transporter presence in plasma membrane. It also results in phosphorylation of SG2NA, zinedin, and catalytical subunit, although co-precipitation, immunocytochemistry, and proximity ligation assay experiments showed that only the amount of SG2NA decreased in the complex with Octn2. PP2A inhibition with okadaic acid does not lead to Octn2 phosphorylation; however, it abolishes observed effects of PKC activation. We postulate that PKC phosphorylates SG2NA, resulting in its dissociation from the complex and transfer of Octn2 to the plasma membrane, leading to increased transporter activity. The observed interaction could affect brain functioning invivo, both in fatty acid metabolism and in control of carnitine homeostasis, known to change in certain brain pathologies.