Vmax For Transport Research Articles

Discovering Palmitoylation Sites on the Dopamine Transporter

The dopamine transporter (DAT) controls synaptic dopamine (DA) levels by reuptake after presynaptic neuronal vesicular release. Parkinson disease, addiction, and ADHD are neurological disorders associated with abnormal DA homeostasis causing DAT dysregulation to be hypothesized as a contributing factor in these disorders. Several binding partner interactions and posttranslational modifications combine to regulate DAT function. One such modification is S‐palmitoylation, the reversible, catalytic addition of palmitate to cysteine via a thioester linkage. S‐palmitoylation can regulate protein‐protein interactions, protein trafficking, transmembrane domain tilting, and membrane stability, and DAT palmitoylation increases DA transport Vmax without altering DAT surface expression and opposes DAT degradation. Because S‐palmitoylation is thought to be an intracellular process, we generated five cytosolic accessible cysteine to alanine mutants at residues Cys6, Cys135, Cys341, Cys522, and Cys580 and analyzed them for palmitoylation by [3H]palmitic acid metabolic labeling. A significant decrease in labeling was seen in only the C580A mutant versus WT control; however, approximately fifty percent of the signal remained, implying the presence of additional S‐palmitoylation site(s). To this end the more sensitive acyl‐biotinyl exchange (ABE) method was used to reanalyze these same mutants, and similar results were obtained confirming Cys580 as a major palmitoylation site with similar palmitoylation reduction in the C580A DAT and approximately 50% remaining residual palmitoylation. To further assess palmitoylation at individual cysteines, all but one of the intracellular cysteines were mutated to alanine, leaving a single cysteine available for palmitoylation. ABE analysis of these mutants showed significantly elevated palmitoylation of DATs containing Cys6 or Cys580 relative to those containing Cys135, Cys341, or Cys522 and mutants containing both Cys6 and Cys580 displayed near WT levels of palmitoylation. To assess if endogenous N‐terminal palmitoylation is present, we performed peptide mapping of rat striatal DAT using endoproteinase Asp‐N digestion prior to ABE and epitope specific immunoblotting. Asp‐N DAT cleavage occurs at Asp174, producing a characteristic 19 kDa peptide fragment containing Cys6 and Cys135 when blotted with a DAT N‐terminal antibody. This analysis revealed palmitoylation of this fragment, indicating one or both of these residues is palmitoylated suggesting further analysis is necessary to distinguish between these sites. Together these results provide evidence supporting DAT palmitoylation at Cys6, implying the potential for tethering of the distal N‐terminus to the plasma membrane. Additionally, Cys6 is adjacent to a known PKC‐mediated phosphorylation site, Ser7, suggesting the potential for interplay between phosphorylation and palmitoylation at these sites and a role for these modifications in DAT regulation.Support or Funding InformationSupported by National Institute on Drug Abuse grants R15 DA031991 (J.D.F.) and R01 DA13147 (R.A.V.) and NIH grants P20GM103442/P20RR016471 (to UND).

Curcumin directly inhibits the transport activity of GLUT1.

Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin's inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin.

Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics

The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders.

Effects of Antiviral Drugs on Organic Anion Transport in Human Placental BeWo Cells.

Placental drug transfer is important for achieving better pharmacotherapy in pregnant women and in fetuses. In the present study, we examined the effects of anti-hepatitis C virus (HCV) and anti-HIV drugs on organic anion transport in human placental BeWo cells. The cellular uptake of two fluorescence organic anions, 8-(2-[fluoresceinyl]aminoethylthio)adenosine-3',5'-cyclic monophosphate (8-FcAMP) and fluorescein, was temperature and concentration dependent. The Michaelis constant (Km) and the maximum uptake rate (Vmax) for 8-FcAMP transport in BeWo cells were estimated to be 6.45 ± 0.75 μM and 25.55 ± 5.93 pmol/mg protein/10 min, respectively. The Km and Vmax values for fluorescein uptake were estimated to be 31.2 ± 11.8 μM and 510.9 ± 90.6 pmol/mg protein/10 min, respectively. Several known substrates of organic anion transporters in human placenta, including atorvastatin, glibenclamide, estrone-3-sulfate, and rifampin, inhibited cellular uptake of 8-FcAMP and fluorescein in BeWo cells. Transport of 8-FcAMP and fluorescein was inhibited by the antiviral drugs boceprevir, telaprevir, elvitegravir, and maraviroc. These findings suggest that some antiviral drugs are sufficiently potent to influence placental drug transfer and cause drug-drug interactions.

P.1.e.008 A role of adenylate cyclase 8 in stress-induced behaviour?

Past research has indicated that chronic ethanol exposure enhances dopamine (DA) neurotransmission in several brain regions. The present study examined the effects of chronic ethanol drinking on dopamine transporter (DAT) function in the nucleus accumbens (Acb) of High-Alcohol-Drinking replicate line 1 (HAD-1) rats. HAD rats were given concurrent 24-h access to 15% ethanol and water or water alone for 8 weeks. Subsequently, DA uptake and the Vmax of the DAT were compared between the two groups using homogenates of the nucleus accumbens. DA uptake was measured following a 2 min incubation at 37°C in the presence of 8 nM [3H]DA. For kinetic analyses, DA uptake was assessed in the presence of 5 concentrations of [3H]DA ranging from 8 nM to 500 nM. Analyses of the data revealed a significant increase in DA uptake in the ethanol group compared to water controls. Kinetic analyses revealed the change in DA uptake to be a consequence of an increase in the Vmax of transport. These findings demonstrate that chronic free-choice oral ethanol consumption in HAD-1 female rats increases DA uptake in the Acb by increasing the Vmax of the transporter. However, it is not known whether the ethanol-induced change in Vmax is caused by differences in the actual number of available transporter sites or from a difference in the velocity of operation of a similar number of transporters. Overall, the data indicate that chronic ethanol consumption by HAD-1 rats produces prolonged neuroadaptations within the mesolimbic DA system, which may be important for the understanding of the neurobiological basis of alcoholism.

Acetaminophen (AP) inhibits intestinal P‐glycoprotein (P‐gp) activity in vitro and in vivo.

AP induces long‐term regulation of expression of intestinal P‐gp. Whether it can acutely modulate P‐gp activity is unknown.AimsTo study the effect of AP on rat intestinal P‐gp activity in vivo and in vitro.M&MIn vitro. Intestinal everted sacs were filled with rhodamine 123 (serosal), a tipical P‐gp substrate, (R123; 4.5; 9; 12; 18 or 36 μM) and incubated in KH buffer (mucosal) alone or with AP (100 μM), and secretion of R123 was assessed along a 40‐min period. Serosal‐mucosal transport of AP was studied in a similar fashion, in the presence/absence of Psc 833 (10 μM), a specific inhibitor of P‐gp. Transport of AP (10 μM) was also evaluated in P‐gp knock down HepG2 cells. In vivo. Intestinal absorption of digoxin (Dig, 25.6 nmol/kg) was studied by portal vein sampling (30–min period) in the presence or absence of AP (100 μM).ResultsR123 transport adjusted well to a sigmoideal curve. Its Vmax was decreased by AP (P<0.05), with no modification in EC50 or slope. AP transport was not affected by PSC 833 or by knocking down P‐gp. In vivo intestinal absorption of Dig was increased (+214%) by AP (P<0.05).ConclusionAP inhibited P‐gp activity in vitro and in vivo. AP‐induced changes in Vmax of R123 transport, together with lack of AP transport by P‐gp, suggest that inhibition is unlikely competitive. Bioavailability and therapeutic efficacy of drugs, substrates of P‐gp, can be altered when they are co‐administered with AP.

Radiotracers for cardiac sympathetic innervation: Transport kinetics and binding affinities for the human norepinephrine transporter

Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [(11)C]-(-)-meta-hydroxyephedrine, [(11)C]-(-)-epinephrine, and a series of [(11)C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [(3)H]-labeled biogenic amines were also determined. Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [(3)H]mazindol provided estimates of binding affinities (KI) for NET. Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for 'tracer concentrations' of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r(2)=0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density.

The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal

Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity. We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1. These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.

Syntaxin 1C, a soluble form of syntaxin, attenuates membrane recycling by destabilizing microtubules

Syntaxin 1C (STX1C), produced by alternative splicing of the stx1A gene, is a soluble syntaxin lacking a SNARE domain and a transmembrane domain. It is unclear how soluble syntaxin can control intracellular membrane trafficking. We found that STX1C affected microtubule (MT) dynamics through its tubulin-binding domain (TBD) and regulated recycling of intracellular vesicles carrying glucose transporter-1 (GLUT1). We demonstrated that the amino acid sequence VRSK of the TBD was important for the interaction between STX1C and tubulin and that wild-type STX1C (STX1C-WT), but not the TBD mutant, reduced the V(max) of glucose transport and GLUT1 translocation to the plasma membrane in FRSK cells. Moreover, by time-lapse analysis, we revealed that STX1C-WT suppressed MT stability and vesicle-transport motility in cells expressing GFP-α-tubulin, whereas TBD mutants had no effect. We also identified that GLUT1 was recycled in the 45 minutes after endocytosis and that GLUT1 vesicles moved along with MTs. Finally, we showed, by a recycling assay and FCM analysis, that STX1C-WT delayed the recycling phase of GLUT1 to PM, without affecting the endocytotic process of GLUT1. These data indicate that STX1C delays the GLUT1 recycling phase by suppressing MT stability and vesicle-transport motility through its TBD, providing the first insight into how soluble syntaxin controls membrane trafficking.

Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

Glycosylation of the OCTN2 carnitine transporter: Study of natural mutations identified in patients with primary carnitine deficiency

Primary carnitine deficiency is caused by impaired activity of the Na +-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.

Syntaxin 1A regulates dopamine transporter activity, phosphorylation and surface expression

We investigated the functional relationship between the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin 1A (syn 1A) and the dopamine transporter (DAT) by treating rat striatal tissue with Botulinum Neurotoxin C (BoNT/C) and co-transfecting syn 1A with DAT in non-neuronal cells, followed by analysis of DAT activity, phosphorylation, and regulation. Treatment of striatal slices with BoNT/C resulted in elevated dopamine (DA) transport Vmax and reduced DAT phosphorylation, while heterologous co-expression of syn 1A led to reduction in DAT surface expression and transport Vmax. Syn 1A was present in DAT immunoprecipitation complexes, supporting a direct or indirect interaction between the proteins. Phorbol ester regulation of DA transport activity was retained in BoNT/C-treated synaptosomes and syn 1A transfected cells, demonstrating that protein kinase C (PKC) and syn 1A effects occur through independent processes. These findings reveal a novel mechanism for regulation of DAT activity and phosphorylation, and suggest the potential for syn 1A to impact DA neurotransmission through effects on reuptake.

2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases

By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20mM) than for the wild (3-6mM) cells. Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5-8 pH range. More differences in protein content (37-64mgg(-1) cell dry weight) and number of resolved spots (178-307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species.

Single nucleotide polymorphisms in the human Na+-dicarboxylate cotransporter affect transport activity and protein expression

The sodium-coupled transport of citric acid cycle intermediates in the intestine and kidney is mediated by the Na(+)-dicarboxylate cotransporter, NaDC1. In the kidney, NaDC1 plays an important role in regulating succinate and citrate concentrations in the urine, which may have physiological consequences including the development of kidney stones. In the present study, the impact of nonsynonymous single nucleotide polymorphisms (SNPs) on NaDC1 expression and function was characterized using the COS-7 cell heterologous expression system. The I550V variant had an increased sensitivity to lithium inhibition although there were no significant effects on protein abundance. The L44F variant had no significant effects on expression or function. The membrane protein abundance of the M45L, V117I, and F254L variants was decreased, with corresponding decreases in transport activity. The A310P variant had decreased protein abundance as well as a change in substrate selectivity. The P385S variant had a large decrease in succinate transport V(max), as well as altered substrate selectivity, and a change in the protein glycosylation pattern. The most damaging variant was V477M, which had decreased affinity for both succinate and sodium. The V477M variant also exhibited stimulation by lithium, indicating a change in the high-affinity cation binding site. We conclude that most of the naturally occurring nonsynonymous SNPs affect protein processing of NaDC1, and several also affect functional properties. All of these mutations are predicted to decrease transport activity in vivo, which would result in decreased intestinal and renal absorption of citric acid cycle intermediates.

Homocysteine Methyltransferases Mht1 and Sam4 Prevent the Accumulation of Age-damaged (R,S)-AdoMet in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosyl-l-methionine (AdoMet) is spontaneously degraded by inversion of its sulfonium center to form the R,S diastereomer. Unlike its precursor, (S,S)-AdoMet, (R,S)-AdoMet has no known cellular function and may have some toxicity. Although the rate of (R,S)-AdoMet formation under physiological conditions is significant, it has not been detected at substantial levels in vivo in a wide range of organisms. These observations imply that there are mechanisms that either dispose of (R,S)-AdoMet or convert it back to (S,S)-AdoMet. Previously, we identified two homocysteine methyltransferases (Mht1 and Sam4) in yeast capable of recognizing and metabolizing (R,S)-AdoMet. We found similar activities in worms, plants, and flies. However, it was not established whether these activities could prevent R,S accumulation. In this work, we show that both the Mht1 and Sam4 enzymes are capable of preventing R,S accumulation in Saccharomyces cerevisiae grown to stationary phase; deletion of both genes results in significant (R,S)-AdoMet accumulation. To our knowledge, this is the first time that such an accumulation of (R,S)-AdoMet has been reported in any organism. We show that yeast cells can take up (R,S)-AdoMet from the medium using the same transporter (Sam3) used to import (S,S)-AdoMet. Our results suggest that yeast cells have evolved efficient mechanisms not only for dealing with the spontaneous intracellular generation of the (R,S)-AdoMet degradation product but for utilizing environmental sources as a nutrient.

Acute Modulation of Sugar Transport in Brain Capillary Endothelial Cell Cultures during Activation of the Metabolic Stress Pathway

GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V(max) for zero-trans 3-O-methylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V(max) for equilibrium exchange transport (intracellular [sugar] = extracellular [sugar]). GLUT1, GLUT8, and GLUT9 mRNAs are detected in bEnd.3 cells where GLUT1 mRNA levels are 33-fold greater than levels of GLUT8 or GLUT9 mRNA. Neither GLUT1 mRNA nor total protein levels are affected by acute metabolic stress. Cell surface biotinylation reveals that plasma membrane GLUT1 levels are increased 2-3-fold by metabolic depletion, although cell surface Na(+),K(+)-ATPase levels remain unaffected by ATP depletion. Treatment with the AMP-activated kinase agonist, AICAR, increases V(max) for net 3-O-methylglucose uptake by 2-fold. Glucose depletion and treatment with potassium cyanide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and AICAR also increase AMP-dependent kinase phosphorylation in bEnd.3 cells. These results suggest that metabolic stress rapidly stimulates blood-brain barrier endothelial cell sugar transport by acute up-regulation of plasma membrane GLUT1 levels, possibly involving AMP-activated kinase activity.

Transmembrane Domain 6 of the Human Serotonin Transporter Contributes to an Aqueously Accessible Binding Pocket for Serotonin and the Psychostimulant 3,4-Methylene Dioxymethamphetamine

The plasma membrane serotonin (5-HT) transporter (SERT, SLC6A4) clears 5-HT after release at nerve termini and is targeted by both antidepressant medications and psychostimulants (e.g. MDMA, cocaine). Homology modeling of human SERT (hSERT), based on high resolution structures of the microbial SLC6 family member LeuT(Aa), along with biochemical studies of wild type and mutant transporters, predicts transmembrane (TM) domains 1, 3, 6, and 8 comprise the 5-HT-binding pocket. We utilized the substituted cysteine accessibility method along with surface and site-specific biotinylation to probe TM6 for aqueous accessibility and differential interactions with 5-HT and psychostimulants. Our results are consistent with TM6 being composed of an aqueous-accessible, alpha-helical extracellular domain (TM6a) that is separated by a central, unwound section from a cytoplasmically localized domain (TM6b) with limited aqueous accessibility. The substitution G338C appears to lock hSERT in an outward-facing conformation that, although accessible to aminoethylmethanethiosulfonate-biotin, 5-HT, and citalopram, is incapable of inward 5-HT transport. Transport of 5-HT by G338C can be partially restored by the TM1 mutation Y95F. With regard to methanethiosulfonate (MTS) inactivation of uptake, TM6a Cys mutants demonstrate Na(+)-dependent [2-(trimethylammonium)ethyl]-MTS sensitivity. Studies with the centrally located substitution S336C reveal features of a common binding pocket for 5-HT and 3,4-methylenedioxymethamphetamine (MDMA). Interestingly, the substitution I333C reveals an MDMA-induced conformation not observed with 5-HT. In the context of prior studies on TM1, our findings document shared and unique features of TM6 contributing to hSERT aqueous accessibility, ligand recognition, and conformational dynamics.

Trans effects of Na + and glucose on Na+/glucose co‐transport

The human intestinal Na+/glucose co‐transporter 1 (SGLT1) is responsible for glucose absorption on the brush border of the small intestine. Previously, it has been shown that two Na+ ions bind to the extracellular surface of SGLT1 prior to sugar binding and translocation. However, the order of release on the cytoplasmic surface is not well understood. Molecular dynamics simulations based on the X‐ray crystal structure of a homolog of SGLT1 suggest that Na+ is the first substrate to dissociate from the cytoplasmic (i.e. trans) side of the transporter during a reaction cycle. To test this, we obtained whole cell patch clamp recordings in human SGLT1‐expressing HEK293T cells with varying trans substrate concentrations. Compared with control experiments under zero trans conditions, 85 mM trans glucose reduced the Vmax for transport by greater than two‐fold. In contrast, trans Na+ alone had no effect on Vmax. These results are consistent with kinetic modeling that predicts sugar is first to dissociate on the cytoplasmic surface of SGLT1. This work was supported by NIH grant #DK19567 and pre‐doctoral fellowship #DK821532 to CSH.

Chemotaxis to ammonium/methylammonium in Chlamydomonas reinhardtii: the role of transport systems for ammonium/methylammonium

Ammonium is the preferred nitrogen source and chemoattractant for the unicellular green alga Chlamydomonas reinhardtii. Here we describe that chemotaxis to methylammonium, a non-metabolizable analogue to ammonium, that occurs only in vegetative cells and pregametes but not in gametes. A new methylammonium-resistant mutant, hat1 that was generated by insertional mutagenesis, has been isolated and found to be affected at multiple loci. At the physiological level, hat1 showed altered Km and Vmax for high-affinity ammonium and methylammonium transport. Transcript levels of the ammonium transporter genes CrAmt1.(1-8) were similar to the wild type except for a lower expression level of CrAmt1.5, 6 and 8. Treatment with the potassium-channel inhibitor tetraethylammonium (TEA) blocked chemotaxis to ammonium/methylammonium and [14C]-methylammonium uptake. Our results suggest that the hat1 mutant could be affected in genes encoding regulatory elements of AMT1 transporter activities and that chemotaxis to ammonium/methylammonium is mediated by ammonium transporters sensitive to the potassium-channel inhibitor TEA.

Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K(m) and V(max) of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.