Plasma Membrane Transport Proteins Research Articles

CsRCI2D enhances high-temperature stress tolerance in Camelina sativa L. through endo-membrane trafficking from the plasma membrane

Rare Cold Inducible 2s (RCI2s) are hydrophobic proteins in cell membranes that participate in abiotic stress tolerance mechanisms. Additionally, they are used as traceable membrane trafficking markers in endocytosis studies. Plants regulate cell homeostasis through endocytosis by limiting the activity of plasma membrane transporter proteins to adapt to stressful conditions. In this study, we found high temperature (HT) stress-induced membrane trafficking of RCI2D in Camelina sativa L. The gene expression and protein synthesis were increased by HT stress at 37 °C. Moreover, rapid membrane trafficking of CsRCI2D was traced by multiple-phase membrane fractionation using sucrose density gradients and compared with CsRCI2E/F/G from the same protein family subgroup. The distribution of CsRCI2s was shown to be similar to that of the clathrin heavy chain, which is known as a major endocytosis protein. Subcellular localization of CsRCI2D was observed in the plasma membrane and endo-membranes and overlapped with membrane lipids. CsRCI2D co-localized with lipids, and its overexpression increased the intracellular lipid content compared to that of wild-type camelina. Moreover, transgenic camelina lines showed enhanced HT stress tolerance during germination and hypocotyl elongation when compared to the wild type. These results suggest that HT-induced CsRCI2D membrane trafficking enhances HT stress tolerance in camelina.

Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca2+ and Na+ Channels.

One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

ABSTRACTThe capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor.

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.

Epigenetic Regulation of the Ontogenic Expression of the Dopamine Transporter.

The dopamine transporter (DAT) is a plasma membrane transport protein responsible for regulating the duration and intensity of dopaminergic signaling. Altered expression of DAT is linked to neurodevelopmental disorders, including attention deficit hyperactivity disorder and autism spectrum disorder, and is shown to contribute to the response of psychotropic drugs and neurotoxicants. Although the postnatal levels of DAT have been characterized, there are few data regarding the mechanisms that regulate postnatal DAT expression. Here, we examine the ontogeny of DAT mRNA from postnatal days 0 to 182 in the rat brain and define a role for epigenetic mechanisms regulating DAT expression. DAT mRNA and protein significantly increased between PND 0 and 6 months in rat midbrain and striatum, respectively. The epigenetic modifiers Dnmt1, Dnmt3a, Dnmt3b, and Hdac2 demonstrated age associated decreases in mRNA expression whereas Hdac5 and Hdac8 showed increased mRNA expression with age. Chromatin immunoprecipitation studies revealed increased protein enrichment of acetylated histone 3 at lysines 9 and 14 and the dopaminergic transcription factors Nurr1 and Pitx3 within the DAT promoter in an age-related manner. Together these studies provide evidence for the role of epigenetic modifications in the regulation of DAT during development. The identification of these mechanisms may contribute to potential therapeutic interventions aimed at neurodevelopmental disorders of the dopaminergic system.

Correlations between striatal dopamine transporter distribution, glucose metabolism and clinical symptoms in Parkinson′s disease

Objective To investigate the correlations among striatal dopamine transporter (DAT) distribution, glucose metabolism and Parkinson′s disease (PD) clinical symptoms. Methods Twenty-five clinically confirmed idiopathic PD patients (17 males, 8 females, age: (59.8±9.2) years) who underwent 11C-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (CFT) and 18F-fluorodeoxyglucose (FDG) PET imaging from January 2015 to December 2016 were reviewed. The detailed clinical scores were systematically collected from all patients. Correlations between DAT distribution, glucose metabolism and clinical symptoms were evaluated at global and voxel levels using Pearson correlation analysis. Results There were significantly positive correlations between the PD-related pattern (PDRP) value and unified PD rating scale (UPDRS) motor scores, non-motor symptoms scale (NMSS) scores, activity of daily living scale (ADL) scores (r values: 0.580, 0.522, 0.557, all P 0.05). The CFT uptake of ipsilateral caudate nucleus and anterior putamen were negatively correlated with ADL scores (r values: -0.502, -0.522, both P 0.05). The presence of the significant correlations between UPDRS motor scores, ADL scores and the CFT radioactive count were confirmed in left caudate nucleus and left putamen (r values: from -0.90 to -0.47, all P<0.05). The metabolic PET imaging disclosed a set of brain regions correlating with the clinical symptoms. The presence of significant correlations between the metabolic PET imaging and CFT uptake were confirmed in bilateral caudate nucleus (r values: 0.47-0.90, both P<0.01), precentral gyrus and insula (r values: -0.90 to -0.47, all P<0.01). Conclusion The correlations between DAT distribution, glucose metabolism and PD clinical symptoms are complicated, which promote the understanding in the proper application of dopaminergic and metabolic PET imaging in PD and offer more evidences in PD pathophysiological mechanisms. Key words: Parkinson disease; Dopamine plasma membrane transport proteins; Signs and symptoms; Positron-emission computed; Deoxyglucose

Structural and Functional Changes in the Na+/H+ Exchanger Isoform 1, Induced by Erk1/2 Phosphorylation.

The human Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane transport protein that plays an important role in pH regulation in mammalian cells. Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, protons accumulate within the cytosol. Extracellular signal-regulated kinase (ERK)-mediated regulation of NHE1 is important in several human pathologies including in the myocardium in heart disease, as well as in breast cancer as a trigger for growth and metastasis. NHE1 has a N-terminal, a 500 amino acid membrane domain, and a C-terminal 315 amino acid cytosolic domain. The C-terminal domain regulates the membrane domain and its effects on transport are modified by protein binding and phosphorylation. Here, we discuss the physiological regulation of NHE1 by ERK, with an emphasis on the critical effects on structure and function. ERK binds directly to the cytosolic domain at specific binding domains. ERK also phosphorylates NHE1 directly at multiple sites, which enhance NHE1 activity with subsequent downstream physiological effects. The NHE1 cytosolic regulatory tail possesses both ordered and disordered regions, and the disordered regions are stabilized by ERK-mediated phosphorylation at a phosphorylation motif. Overall, ERK pathway mediated phosphorylation modulates the NHE1 tail, and affects the activity, structure, and function of this membrane protein.

Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog.

Membrane traffic plays a pivotal role in virulence in the enteric protozoan parasite Entamoeba histolytica. EhRab8A small GTPase is a key regulator of membrane traffic at the endoplasmic reticulum (ER) of this protist and is involved in the transport of plasma membrane proteins. Here we identified the binding proteins of EhRab8A. The Cdc50 homolog, a non-catalytic subunit of lipid flippase, was identified as an EhRab8A binding protein candidate by affinity coimmunoprecipitation. Binding of EhRab8A to EhCdc50 was also confirmed by reciprocal immunoprecipitation and blue-native polyacrylamide gel electrophoresis, the latter of which revealed an 87 kDa complex. Indirect immunofluorescence imaging with and without Triton X100 showed that endogenous EhCdc50 localized on the surface in the absence of permeabilizing agent but was observed on the intracellular structures and overlapped with the ER marker Bip when Triton X100 was used. Overexpression of N-terminal HA-tagged EhCdc50 impaired its translocation to the plasma membrane and caused its accumulation in the ER. As reported previously in other organisms, overexpression and accumulation of Cdc50 in the ER likely inhibited surface transport and function of the plasma membrane lipid flippase P4-ATPase. Interestingly, HA-EhCdc50-expressing trophozoites gained resistance to miltefosine, which is consistent with the prediction that HA-EhCdc50 overexpression caused its accumulation in the ER and mislocalization of the unidentified lipid flippase. Similarly, EhRab8A gene silenced trophozoites showed increased resistance to miltefosine, supporting EhRab8A-dependent transport of EhCdc50. This study demonstrated for the first time that EhRab8A mediates the transport of EhCdc50 and lipid flippase P4-ATPase from the ER to the plasma membrane.

Relationship between remifentanil-induced hyperalgesia and function of nitrated glutamate transportor-1 and glutamine synthetase in spinal cord of rats with incisional pain

Objective To investigate the relationship remifentanil-induced hyperalgesia and function of nitrated glutamate transportor-1 (GLT-1) and glutamine synthetase (GS) in the spinal cord of rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 260-280 g, aged 2-3 months, in which caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), incisional pain group (group I), remifentanil group (group R) and remifentanil plus incisional pain group (group RI). Normal saline was intravenously infused for 60 min at 0.1 ml·kg-1·min-1 in group C. The model of incisional pain was established, and normal saline was simultaneously infused for 60 min via the tail vein at 0.1 ml·kg-1·min-1 in group I. Remifentanil was infused for 60 min via the tail vein at 1.0 μg· kg-1·min-1 in group R. The model of incisional pain was established, and remifentanil was infused for 60 min via the tail vein at 1.0 μg· kg-1·min-1 in group RI.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline (T0) and at 2, 6, 24 and 48 h after infusion (T1-4). The rats were sacrificed after the last measuremnet of pain threshold, and the L4-6 segment of the spinal cord was removed for determination of the expression of GLT-1 and GS (by Western blot) and expression of nitrated GLT-1 (nGLT-1) and nitrated GS (nGS) (by Western blot). Ratios of nGLT-1/GLT-1 and nGS/GS were calculated. Results Compared with group C, the MWT was significantly decreased and TWL was shortened at T1-4, the expression of GLT-1 and GS was down-regulated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1/GLT-1 and nGS/GS were increased in I, R and RI groups (P<0.05). Compared with group I and group R, the MWT was significantly decreased and TWL was shortened at T1-4, the expression of GLT-1 and GS was down-regulated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1/GLT-1 and nGS/GS were increased in group RI (P<0.05). Conclusion The mechanism of remifentanil-induced hyperalgesia may be related to impaired function of GLT-1 and GS in the spinal cord of rats with incisional pain. Key words: Piperidines; Hyperalgesia; Pain, postoperative; Glutamate plasma membrane transport proteins; Glutamate-ammonia ligase; Spinal cord

The loop diuretics bumetanide and ethacrynic acid inhibit voltage‐gated calcium and sodium channels in cultured cortical neurons

One of the consequences of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ have been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke‐induced ischemia and acidosis includes the dysregulation of a number of plasma membrane transport proteins, such as increased activity of the sodium‐potassium‐chloride cotransporter 1 (NKCC1). Experiments using the NKCC1 inhibitors, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects the Na+ and Ca2+ overload observed following in vitro ischemia‐acidosis. Fluorometric Ca2+ and Na+ measurements were performed on cultured cortical neurons from embryonic (E18) rats. Both BMN and EA depressed ischemia‐acidosis induced [Ca2+]i overload, but did not appreciably alter [Na+]i increases observed under these conditions. Patch‐clamp electrophysiology was used to measure whole‐cell membrane currents to identify target(s) of BMN and EA, other than the electroneutral NKCC‐1, responsible for the effects of these drugs on [Ca2+]i. Voltage‐gated Ca2+ channels (VGCC) were inhibited by both BMN and EA with half‐maximal inhibitory concentration (IC50) values of 4 μM and 36 μM respectively. However, maximal concentrations of BMN failed to block ~40% of VGCC mediated currents, while EA completely abolished these responses. Similarly, voltage‐gated Na+ channels (VGSC) were blocked by BMN and EA with IC50 values of 13 μM and 30 μM, respectively. In contrast to the VGCC responses, both loop diuretics inhibited VGSC currents by ~80%. Experiments using the VGSC inhibitor, tetrodotoxin, showed that BMN was preferentially inhibiting TTX‐sensitive currents in these cells. Neither BMN nor EA affected currents mediated by acid‐sensing ion channels or ionotropic glutamatergic receptors. Taken together, our data suggest that these two loop diuretics are likely to decrease injury following ischemic stroke by effectively mitigating [Ca2+]i overload in neurons via the inhibition of both voltage‐gated Ca2+ and Na+ channels. Moreover, the depression of ischemia‐acidosis evoked [Ca2+]i overload by these compounds does not appear to be dependent on inhibition of NKCC1. Given that the inhibitory concentrations observed here for VGCC and VGSC are similar to those reported for block of NKCC1 and NKCC2 by these compounds, loop diuretics are likely to be inhibiting these channels at therapeutic doses used in humans. Therefore, blockade of these channels by loop diuretics may also contribute to other clinical effects of the compounds observed in animal studies and in humans.Support or Funding InformationAmerican Heart Association Grant‐In‐Aid, 17GRNT33661273 (JC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of SLC6A4 gene polymorphism on pain sensitivity in patients with lung cancer

Objective To evaluate the effect of SLC6A4 gene polymorphism on pain sensitivity in the patients with lung cancer. Methods A total of 248 patients with pulmonary malignant tumor served as lung cancer group and 104 healthy subjects in the Physical Examination Center of our hospital served as control group.Patients with malignant pulmonary tumor in lung cancer group were further divided into 3 subgroups according to pain score and the three step analgesic ladder recommended by WHO(opioids was used when visual analogue scale [VAS] score≥4 points): painless subgroup, mild pain subgroup and moderate-severe pain subgroup.The consumption of opioids(based on requirement for morphine)within 24 h after pain relief(VAS score≤3 points)after treatment and VAS score before treatment were recorded.Venous blood samples were collected, and the genotypes were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Results There was no significant difference in genotype frequency or allele frequency at rs4795541 and rs3813034 sites between lung cancer group and control group and among the three subgroups(P>0.05). There was no significant difference in VAS score before treatment or requirement for morphine between patients of different genotypes and alleles(P>0.05). There was no significant difference in VAS score before treatment or requirement for morphine between lung cancer patients with medium and low expression of serotonin transporter in 5-HTT-linked polymorphic region. Conclusion SLC6A4 gene polymorphism exerts no effect on pain sensitivity in the patients with lung cancer. Key words: Serotonin plasma membrane transport proteins; Polymorphism, single nucleotide; Neoplasms; Pain threshold

Maize NPF6 Proteins Are Homologs of Arabidopsis CHL1 That Are Selective for Both Nitrate and Chloride.

Nitrate uptake by plant cells requires both high- and low-affinity transport activities. Arabidopsis thaliana nitrate transporter 1/peptide transporter family (NPF) 6.3 is a dual-affinity plasma membrane transport protein that has both high- and low-affinity functions. At-NPF6.3 imports and senses nitrate and is regulated by phosphorylation at Thr-101 (T101). A detailed functional analysis of two maize (Zea mays) homologs of At-NPF6.3 (Zm-NPF6.6 and Zm-NPF6.4) showed that Zm-NPF6.6 was a pH-dependent nonbiphasic high-affinity nitrate-specific transport protein. By contrast, maize NPF6.4 was a low-affinity nitrate transporter with efflux activity. When supplied chloride, NPF6.4 switched to a high-affinity chloride selective transporter, while NPF6.6 had only a low-affinity chloride transport activity. Structural predictions identified a nitrate binding His (H362) in NPF6.6 but not in NPF6.4. Mutation of NPF6.4 Tyr-370 to His (Y370H) resulted in saturable high-affinity nitrate transport activity and nitrate selectivity. Loss of H362 in NPF6.6 (H362Y) eliminated both nitrate and chloride transport. Furthermore, alterations to Thr-104, a conserved phosphorylation site in NPF6.6, resulted in a similar high-affinity nitrate transport activity with increased Km, whereas equivalent changes in NPF6.4 (T106) disrupted high-affinity chloride transport activity. NPF6 proteins exhibit different substrate specificity in plants and regulate nitrate transport affinity/selectivity using a conserved His residue.

Predictive value of hENT1 immunohistochemistry for gemcitabine-treated pancreatic cancer is antibody dependent.

e15746 Background: Pancreatic cancer (PC) continues to have poor outcomes, and biomarkers to guide individualized therapy are needed. Gemcitabine (GEM), a pyrimidine nucleoside, requires plasma membrane transporter proteins to enter cells, and deficiency of the human equilibrative nucleoside transporter 1 (hENT1) protein confers high-level GEM resistance in pre-clinical models. Furthermore, abundance of hENT in PC cells, as determined by immunohistochemistry (IHC), has been variably reported to be associated with improved survival in people with PC treated with GEM. The antibodies (Abs) used for IHC differ in targeted epitope, method of generation, and species of origin. The purpose of this review is to evaluate the contribution of anti-hENT1 Ab choice in finding a link between hENT1 expression and outcomes. Methods: We reviewed the published literature investigating relationships between hENT1 IHC tumor expression and survival in people with PC treated with GEM. Results: Eighteen studies were identified, using five distinct Abs (Table 1). We identified apparent differences in predictive value among Abs (Chi square test p = 0.065). All seven studies using the 10D7G2 Ab reported that high hENT1 expression is associated with significantly better outcomes in GEM-treated patients. Studies that used other anti-hENT1 Abs, in particular SP120 and 11337-1-AP, had lower rates of success. Pooled HRs and additional metrics will be presented. Conclusions: There is significant heterogeneity among studies evaluating the link between hENT1 expression and outcomes. hENT1 IHC with the 10D7G2 Ab is a strong and reproducible predictive marker for improved survival in people with PC treated with GEM. [Table: see text]

Assessment of effects of 7, 8-dihydroxyflavone on the striatum in normal cynomolgus monkeys with 99Tcm-TRODAT-1 SPECT/CT imaging

Objective To assess the effects of 7, 8-dihydroxyflavone (7, 8-DHF) on the striatum (ST) in normal cynomolgus monkeys using 99Tcm-TRODAT-1 imaging. Methods A total of six healthy female cynomolgus monkeys were included in this study. Three of them were fed with normal food (control group), and the other three were given oral administration of 7, 8-DHF in addition to normal food (experimental group). The SPECT/CT imaging was performed at different time after 99Tcm-TRODAT-1 injection. The ROI of ST was drawn on images of 3 consecutive transverse slices that could be visualized best. The cerebellum (CB) was taken as the background reference area. The radioactivity uptake ratios of ST/CB at 1, 3, 4 and 5 h were calculated respectively. Paired-t test was used to analyze the data. Results ST radioactive uptake ratios showed continuing increase on the delay images. ST/CB uptake ratios of the control group at 1, 3, 4 and 5 h were 1.43±0.04, 1.82±0.06, 2.04±0.12, 2.42±0.23, respectively, and those of the experimental group were 1.35±0.08, 2.40±0.09, 2.74±0.13 and 3.25±0.15 respectively. There was no significant difference between the two groups at 1 h (t=2.57, P>0.05), while ST/CB uptake ratios of the experimental group at 3, 4 and 5 h were significantly higher (t values: 2.77, 2.87 and 2.92, all P<0.05). Conclusion 99Tcm-TRODAT-1 SPECT/CT imaging can be used to assess the DAT activation effect by 7, 8-DHF on ST of cynomolgus monkeys. Key words: Corpus striatum; Dopamine plasma membrane transport proteins; Flavones; Tomography, emission-computed, single-photon; Tomography, X-ray computed; Macaca fascicularis; TRODAT-1

VdThit, a Thiamine Transport Protein, Is Required for Pathogenicity of the Vascular Pathogen Verticillium dahliae.

Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. Few plasma-membrane transport proteins for nutrient acquisition have been identified for this fungus, and their involvement in the disease process is unknown. Here, a plasma-membrane protein, the V. dahliae thiamine transporter protein VdThit, was characterized functionally by deletion of the VdThit gene in V. dahliae. Disruption strains were viable, but growth and conidial germination and production were reduced and virulence was impaired. Interestingly, by supplementing exogenous thiamine, growth, conidiation, and virulence of the VdΔThit mutants were partially restored. Stress-tolerance assays showed that the VdΔThit mutant strains were markedly more susceptible to oxidative stress and UV damage. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses showed low levels of pyruvate metabolism intermediates acetoin and acetyl coenzyme A (acetyl-CoA) in the VdΔThit mutant strains, suggesting that pyruvate metabolism was suppressed. Expression analysis of VdThit confirmed the importance of VdThit in vegetative growth, reproduction, and invasive hyphal growth. Furthermore, a green fluorescent protein (GFP)-labeled VdΔThit mutant (VdΔThit-7-GFP) was suppressed in initial infection and root colonization, as viewed with light microscopy. Together, these results showed that VdThit plays an indispensable role in the pathogenicity of V. dahliae.

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy-Causing Mutants of SLC4A11.

Protein misfolding, causing retention of nascent protein in the endoplasmic reticulum (ER), is the most common molecular phenotype for disease alleles of membrane proteins. Strategies are needed to identify therapeutics able to correct such folding/trafficking defects. Mutations of SLC4A11, a plasma membrane transport protein of the human corneal endothelial cell layer, cause cases of congenital hereditary endothelial dystrophy, Harboyan syndrome, and Fuchs' endothelial corneal dystrophy. Most SLC4A11 mutations induce SLC4A11 misfolding and retention in the ER. An assay amenable to high-throughput screening was developed to quantify SLC4A11 at the plasma membrane, enabling a search for potential traffic-correcting small molecules. The assay was validated by comparing cell surface abundance of SLC4A11 mutants measured in the assay to observations from confocal immunofluorescence and values from cell surface biotinylation. Functionality of mutant proteins was assessed, using a confocal microscopic green fluorescent protein (GFP) water flux assay where relative rates of cell swelling are compared. A small-scale screen revealed that the nonsteroidal anti-inflammatory drugs (NSAIDs), glafenine, ibuprofen, and acetylsalicylic acid dissolved in 0.2% dimethyl sulfoxide (DMSO), partially rescued the trafficking defect in some SLC4A11 mutants, expressed in HEK293 cells. These SLC4A11 mutants retained functional activity when rescued to the plasma membrane by glafenine treatment. Glafenine was effective with an EC50 of 1.5 ± 0.7 μM. These data suggest that glafenine, and perhaps other NSAIDs, hold potential as therapeutics for misfolded membrane proteins, like SLC4A11. The high throughput approach described here can be modified to identify correctors of other misfolded plasma membrane proteins that cause eye disease.

Structure of a eukaryotic SWEET transporter in a homotrimeric complex.

Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs. SWEETs carry mono- and disaccharides across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion, phloem loading for long distance translocation, pollen nutrition, and seed filling. Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells. The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection. Here we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice (Oryza sativa), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Insights into the structure-function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.

Association between norepinephrine transporter gene polymorphism and decision-making processing in patients with cerebral infarction

Objective To investigate the association between norepinephrine transporter (NET) gene polymorphism and decision-making processing in patients with cerebral infarction. Methods A total of 145 patients with cerebral infarction and 188 normal controls were enrolled in our study. In all subjects, the polymerase chain reaction (PCR) for NET-T182C/G1287A polymorphism assay, gel electrophoresis, image analysis and enzymatic reaction, gene sequencing methods were used. The relationships of NET-T182C/G1287A genotypes and alleles with decision-making processing were analyzed in patients with cerebral infarction.All participants completed six kinds of choice situational problems. Results There were significant differences in genotype and allele frequencies of T182C and G1287A polymorphism in NET between the patients with cerebral infarction and control group(for NET-T182C: genotype, χ2=4.437, P=0.049, allele frequency, χ2=4.363, P=0.037, OR=0.625, 95%CI: 0.436-0.895; for NET-G1287A: genotype, χ2=8.435, P=0.038, allele frequency, χ2=2.765, P=0.036, OR=1.520, 95%CI: 1.053-2.193). The cerebral infarction patients with three NET-T182C genotypes and T/C alleles all completed six choice scenarios, and the scheme selection probability had no significant difference (all P>0.05). In high-risk and no-risk loss situation (scenario 4), the scheme selection probability had significant difference in cerebral infarction patients with NET-G1287A genotypes and G/A alleles (P 0.05). Conclusions NET-G1287A polymorphism may be associated with decision-making processing in patients with cerebral infarction. In the high-risk and no-risk loss condition, patients with GG genotype and G allele have more loss risk aversion. Key words: Brain infarction; Choice behavior; Norepinephrine plasma membrane transport proteins; Polymorphism, single nucleotide

Effects of sinomenine on expression of glutamate transporter-1 mRNA and glutamate aspartate transporter mRNA in spinal cord of rats with neuropathic pain

Objective To evaluate the effectsof sinomenine on the expression of glutamate transporter-1 (GLT-1) mRNA and glutamate aspartate transporter (GLAST) mRNA in the spinal cord of rats with neuropathic pain (NP).Methods Seventy-two male Wistar rats,weighing 180-220 g,aged 6-8 weeks,were divided into 3 groups (n =24 each) using a random number table:sham operation group (group S); NP group; sinomenine group (group SIN).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 350 mg/kg.NP was induced by chronic constrictive injury.The right sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread.In group S,the right sciatic nerves were only exposed,but not ligated.In group SIN,sinomenine injection 40 mg/kg was injected intraperitoneally once a day starting from the end of operation until 1 day before the animals were sacrificed,while the equal volume of normal saline was given instead in S and NP groups.Mechanical paw withdrawal threshold to von Frey stimuli (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL) were measured at 1 day before operation and 1,3,7 and 14 days after operation (T1-4).The rats were sacrificed after measurement of pain threshold,and their lumbar segments (L4-6) of the spinal cord were immediately removed for determination of GLT-1 mRNA and GLAST mRNA expression in the spinal dorsal horn (by real-time fluorescent quantitative PCR).Results Compared with group S,MWT was significantly decreased and TWL was shortened at T1-4 in NP and SIN groups,the expression of GLT-1 mRNA and GLAST mRNA was up-regulated at T1.2 in group NP,and the expression of GLT-1 mRNA and GLAST mRNA was up-regulated at T1-3 in group SIN.Compared with group NP,MWT was significantly increased,TWL was prolonged,and the expression of GLT-1 mRNA and GLAST mRNA was up-regulated at T1.3 in group SIN.Conclusion Sinomenine can mitigate NP in rats and up-regulation of GLT-1 mRNA and GLAST mRNA expression is involved in the mechanism. Key words: Sinomenium; Neuralgia; Glutamate plasma membrane transport proteins; Spinal cord

Interactions of ion transporters and channels with cancer cell metabolism and the tumour microenvironment

Major changes in intra- and extracellular pH homoeostasis are shared features of most solid tumours. These changes stem in large part from the metabolic shift of most cancer cells towards glycolytic metabolism and other processes associated with net acid production. In combination with oncogenic signalling and impact from factors in the tumour microenvironment, this upregulates acid-extruding plasma membrane transport proteins which maintain intracellular pH normal or even more alkaline compared with that of normal cells, while in turn acidifying the external microenvironment. Mounting evidence strongly indicates that this contributes significantly to cancer development by favouring e.g. cancer cell migration, invasion and chemotherapy resistance. Finally, while still under-explored, it seems likely that non-cancer cells in the tumour microenvironment also exhibit altered pH regulation and that this may contribute to their malignant properties. Thus, the physical tumour microenvironment and the cancer and stromal cells within it undergo important reciprocal interactions which modulate the tumour pH profile, in turn severely impacting on the course of cancer progression. Here, we summarize recent knowledge of tumour metabolism and the tumour microenvironment, placing it in the context of tumour pH regulation, and discuss how interfering with these properties may be exploited clinically.