Modulation Of Transporter Activity Research Articles

Ion and lipid orchestration of secondary active transport.

Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondaryactive transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.

Targeting multidrug resistance-associated protein 1 (MRP1)-expressing cancers: Beyond pharmacological inhibition.

Resistance to chemotherapy remains one of the most significant obstacles to successful cancer treatment. While inhibiting drug efflux mediated by ATP-binding cassette (ABC) transporters is a seemingly attractive and logical approach to combat multidrug resistance (MDR), small molecule inhibition of ABC transporters has so far failed to confer clinical benefit, despite considerable efforts by medicinal chemists, biologists, and clinicians. The long-sought treatment to eradicate cancers displaying ABC transporter overexpression may therefore lie within alternative targeting strategies. When aberrantly expressed, the ABC transporter multidrug resistance-associated protein 1 (MRP1, ABCC1) confers MDR, but can also shift cellular redox balance, leaving the cell vulnerable to select agents. Here, we explore the physiological roles of MRP1, the rational for targeting this transporter in cancer, the development of small molecule MRP1 inhibitors, and the most recent developments in alternative therapeutic approaches for targeting cancers with MRP1 overexpression. We discuss approaches that extend beyond simple MRP1 inhibition by exploiting the collateral sensitivity to glutathione depletion and ferroptosis, the rationale for targeting the shared transcriptional regulators of both MRP1 and glutathione biosynthesis, advances in gene silencing, and new molecules that modulate transporter activity to the detriment of the cancer cell. These strategies illustrate promising new approaches to address multidrug resistant disease that extend beyond the simple reversal of MDR and offer exciting routes for further research.

Molecular Determinants for Nitric Oxide Regulation of the Murine Cationic Amino Acid Transporter CAT-2A.

Cationic amino acid transporters (CATs) supply cells with essential and semiessential dibasic amino acids. Among them, l-arginine is the substrate for nitric oxide synthases (NOS) to produce nitric oxide (NO), a key signaling molecule and second messenger. In cardiac preparations, we showed that NO acutely and directly modulates transport activity by noncompetitively inhibiting these CATs. We hypothesize that this NO regulation occurs through modification of cysteine residues in CAT proteins. Homology modeling and a computational chemistry approach identified Cys347 as one of two putative targets for NO binding, of 15 Cys residues present in the low-affinity mouse CAT-2A (mCAT-2A). To test this prediction, mammalian cell lines overexpressing mCAT-2A were used for site-directed mutagenesis and uptake studies. When Cys347 was replaced with alanine (Cys347Ala), mCAT-2A became insensitive to inhibition by NO donors. In addition, the transport capacity of this variant decreased by >50% compared to that of the control, without affecting membrane expression levels or apparent affinities for the transported amino acids. Interestingly, replacing Cys347 with serine (Cys347Ser) restored uptake levels to those of the control while retaining NO insensitivity. Other Cys residues, when replaced with Ala, still produced a NO-sensitive CAT-2A. In cells co-expressing NOS and mCAT-2A, exposure to extracellular l-arginine inhibited the uptake activity of control mCAT-2A, via NO production, but not that of the Cys347Ser variant. Thus, the -SH moiety of Cys347 is largely responsible for mCAT-2A inhibition by NO. Because of the endogenous NO effect, this modulation is likely to be physiologically relevant and a potential intervention point for therapeutics.

Environmental contaminants modulate transport activity of zebrafish organic anion transporters Oat1 and Oat3

Organic anion transporters (OATs) are transmembrane proteins which belong to SLC22 subfamily. They are responsible for the uptake of various endo- and xenobiotics into the cells of different organs and tissues. Following our previous work on characterization of zebrafish Oat1 and Oat3, in this study we analyzed interaction of various classes of environmental contaminants with these membrane transporters using the transport activity assay with HEK293 Flp-In cell line stably overexpressing zebrafish Oat1 and Oat3, respectively. Based on the initial screening of a series of 36 environmental contaminants on their ability to interact with zebrafish Oat1 and Oat3, the most potent interactors were selected, their IC50 values calculated and type of interaction determined. Finally, to further confirm the type of interaction and initially evaluate their toxic potential, the cytotoxicity assays were performed. Broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs was confirmed and potent interactors among environmental contaminants identified.

P-Glycoprotein Activity Is Non-Monotonically Modulated by Transmembrane Voltage

P-glycoprotein (Pgp) is a broadly polyspecific exporter of hydrophobic and amphiphilic compounds that plays an important role in the blood-brain barrier and in multidrug resistance in cancer. Several research groups have recently made significant strides toward elucidating the mechanism of Pgp's catalytic cycle, but the biophysical properties of the cellular microenvironment can have strong effects on the function of Pgp and other ATP-binding cassette (ABC) transporters in as-yet poorly defined ways. Here we report that Pgp, MRP1, and BCRP, three of the most clinically relevant multidrug resistance-linked ABC transporters, are strongly and non-monotonically regulated by the transmembrane electrical potential difference. We confirmed this result with several assays, which used fluorescence and electrophysiological methods to measure the efflux of transporter substrates from two human cell lines and giant unilamellar vesicles and controlled the voltage using electrolyte gradients or the patch clamp technique. At hyperpolarized (highly inside-negative) potentials, each transporter displayed a pronounced reduction in efflux activity compared to the activity at the resting potential; Pgp and MRP1 also showed reduced activity at inside-positive potentials. The transport data is well-described by a model in which the transmembrane potential biases the conformational distribution of the proteins in a manner similar to the mechanism of action of voltage-gated ion channels. In this model, which is compatible with the “alternating access” framing of Pgp function, each catalytic cycle contains two transitions with an opposite dependence on the transmembrane voltage due to the movement of an equivalent of 3-4 elementary charges back and forth across the membrane. This finding, which points to an additional axis for modulating transporter activity, underscores the importance of electric field effects on the function of all membrane proteins, particularly those that undergo large conformational changes in the transmembrane region.

Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters

1. Organophosphorus pesticides (OPs) are known to interact with human ATP-binding cassette drug efflux pumps. The present study was designed to determine whether they can also target activities of human solute carrier (SLC) drug transporters.2. The interactions of 13 OPs with SLC transporters involved in drug disposition, such as organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs), were mainly investigated using transporter-overexpressing cell clones and fluorescent or radiolabeled reference substrates.3. With a cut-off value of at least 50% modulation of transporter activity by 100 µM OPs, OAT1 and MATE2-K were not impacted, whereas OATP1B1 and MATE1 were inhibited by two and three OPs, respectively. OAT3 activity was similarly blocked by three OPs, and was additionally stimulated by one OP. Five OPs cis-stimulated OATP2B1 activity. Both OCT1 and OCT2 were inhibited by the same eight OPs, including fenamiphos and phosmet, with IC50 values however in the 3–30 µM range, likely not relevant to environmental exposure.4. These data demonstrated that various OPs inhibit SLC drug transporter activities, especially those of OCT1 and OCT2, but only when used at high concentrations not expected to occur in environmentally-exposed humans.

Computational modelling of placental amino acid transfer as an integrated system

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia.

The influence of soybean extract on the expression level of selected drug transporters, transcription factors and cytochrome P450 genes encoding phase I drug-metabolizing enzymes.

Soybean phytoestrogens, such as genistein and daidzein, reduce climacteric symptoms and the risk of certain chronic diseases such as cancer and cardiovascular diseases. Despite their widespread use in functional foods and dietary supplements, there is very little data available on their safety and herb-drug interactions, especially with antineoplastic agents. Hence, the aim of our study was to assess the effects of soybean extracts on the expression level of CYP genes and their transcriptional factors. We also investigated the effect of soybean on the mRNA level of transporters, such as P-glycoprotein (MDRI) and multidrug resistance-associated proteins (MRP1, MRP2). Wistar rats were fed a standardized soybean extract (100 mg/kg, p.o.). cDNA was synthesized from total RNA isolated from different tissues (liver and intestinal epithelium) using reverse transcription. Gene expression level was analyzed by RT-PCR method. We demonstrated a significant increase of CYP1A1 mRNA level (by 89%, p = 0.002 and 125%, p = 0.004) as compared with the control group. An increase of AHR and CAR expression after 10 days was also observed (by 60%, p < 0.001 and 52%, p > 0.05, respectively). Additionally an inductive effect for CYP2D1 by 22% (p = 0.008), Mdr1a by 267% (p < 0.0001), Mdr2b by 86% (p < 0.00001), Mrp1 by 9-fold (p < 0.0001), Mrp2 by 83% (p < 0.0001) in the liver and for Mrp2 by 35% (p < 0.001) in the intestinal epithelium, was evaluated. A significant decrease of mRNA level was observed for CYP3A1 (human CYP3A4) in the liver and Mdr1b in the intestinal epithelium. Moreove, we also showed a slight decrease in the amount of mRNA for CAR, PXR and ARNT after 3 days. Our results suggest that Glycine max may change the expression level of CYPs, especially CYP3A4 and CYP1A 7, involved in biotransformation of xenobiotics (drugs, procarcinogens) and may participate in clinically significant interactions with drugs metabolized by these enzymes. Moreover an increase of CYP1A1 (homologue to human CYPIA 1) mRNA level may not only reduce the carcinogenicity of foreign compounds, but may also activate some compounds to their carcinogenicity In case of transporters, it is considered that an increase of their expression in the body may lead to increased fetoprotection. Also, it may reduce both, the exposure of sensitive tissues (e.g. brain, placenta) to xenobiotics and treatment effectiveness of certain diseases. Hence, the search for a safe substance that could effectively modulate transporter activity especially in the treatment of certain hormone -dependent disorders, e.g. osteoporosis and breast cancer occurring mainly in postmenopausal period, continues.

Mouse organic cation transporter 1 determines properties and regulation of basolateral organic cation transport in renal proximal tubules

The proximal tubule of mouse kidney expresses mouse organic cation transporter 1 (mOCT1), mOCT2, and much less mOCT3. Therefore, mOCT-mediated transport across the basolateral membrane of proximal tubules reflects properties of at least mOCT1 and mOCT2. Here, we unraveled substrate affinities and modulation of transport activity by acute regulation by protein kinases on mOCT1 and mOCT2 separately and compared these findings with those from isolated proximal tubules of male and female mOCT2−/− mice. These data are also compared to our recent reports on isolated tubules from wild-type and mOCT1/2 double knockout (mOCT1/2−/−) mice. OCT-mediated transport in proximal tubules of mOCT2−/− mice was only 20 % lower compared to those isolated from wild-type mice. While mOCT1 was regulated by all five pathways examined [protein kinase A (PKA), protein kinase C (PKC), p56lck, phosphoinositide 3-kinase (PI3K), and calmodulin (CaM)], mOCT2 activity was modulated by PKA, p56lck, and CaM only, however, in the same direction. As mOCT-mediated transport across the basolateral membrane of mOCT2−/− mice expressing only mOCT1 and to a small amount mOCT3 was identical to that observed for tubules isolated from wild-type mice and to that observed for human embryonic kidney 293 (HEK293) cells stably expressing mOCT1, mOCT1 represents the relevant paralog for OCT-dependent organic cation transport in the mouse kidney. Gender does not play a major role in expression and activity of renal OCT-mediated transport in the mouse. Properties of mouse OCT considerably differ from those of rat or human origin, and thus, observations made in these rodents cannot directly be transferred to the human situation

Changes in the expression of the glutamate transporter EAAT3/EAAC1 in health and disease

Excitatory amino acid transporters (EAATs) are high-affinity Na(+)-dependent carriers of major importance in maintaining glutamate homeostasis in the central nervous system. EAAT3, the human counterpart of the rodent excitatory amino acid carrier 1 (EAAC1), is encoded by the SLC1A1 gene. EAAT3/EAAC1 is ubiquitously expressed in the brain, mostly in neurons but also in other cell types, such as oligodendrocyte precursors. While most of the glutamate released in the synapses is taken up by the "glial-type" EAATs, EAAT2 (GLT-1 in rodents) and EAAT1 (GLAST), the functional role of EAAT3/EAAC1 is related to the subtle regulation of glutamatergic transmission. Moreover, because it can also transport cysteine, EAAT3/EAAC1 is believed to be important for the synthesis of intracellular glutathione and subsequent protection from oxidative stress. In contrast to other EAATs, EAAT3/EAAC1 is mostly intracellular, and several mechanisms have been described for the rapid regulation of the membrane trafficking of the transporter. Moreover, the carrier interacts with several proteins, and this interaction modulates transport activity. Much less is known about the slow regulatory mechanisms acting on the expression of the transporter, although several recent reports have identified changes in EAAT3/EAAC1 protein level and activity related to modulation of its expression at the gene level. Moreover, EAAT3/EAAC1 expression is altered in pathological conditions, such as hypoxia/ischemia, multiple sclerosis, schizophrenia, and epilepsy. This review summarizes these results and provides an overall picture of changes in EAAT3/EAAC1 expression in health and disease.

Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles

Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo, but their overall composition on axonal vesicles and whether this composition directly modulates transport activity are unknown. Here we characterize the intracellular transport and steady-state motor subunit composition of mammalian prion protein (PrP(C)) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrP(C) vesicle motor complexes and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model wherein PrP(C) vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.

Design, function and structure of a monomeric ClC transporter.

Channels and transporters of the CLC family bring about transmembrane movement of inorganic anions in service of a variety of biological tasks, from the arcane - generating the kilowatt pulses by which electric fish parboil their prey - to the quotidian - acidification of endosomes, vacuoles, and lysosomes1. The homodimeric architecture of CLC proteins (Fig 1), initially inferred from single-molecule studies of an elasmobranch Cl− channel2 and later confirmed by crystal structures of bacterial Cl−/H+ antiporters3,4, appears to be universal. Moreover, the basic machinery enabling ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl− and H+ antiport in the transporters - are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial CLC transporter strait-jacketed by covalent crosslinks across the dimer interface and the behavior of a concatameric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits5,6. However, this evidence is only inferential, and since examples are known in which quaternary rearrangements of extramembrane CLC domains that contribute to dimerization modulate transport activity7, we cannot declare as definitive a “parallel pathways” picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric CLC. In this study, we exploit the known structure of a CLC Cl−/H+ exchanger, CLC-ec1 from E. coli, to design mutants that destabilize the dimer interface while preserving both structure and transport function of individual subunits. The results demonstrate that the CLC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl−/H+ exchange in CLC transporters.

Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.

Close encounters of the oily kind: regulation of transporters by lipids.

Neurotransmitter transporters are membrane proteins that serve as key regulators of extracellular neurotransmitter concentrations and have been long viewed as important targets for drug development by the pharmaceutical industry. Although many cellular signaling systems are known to modulate transport activity, much less is known about how transporters communicate with and are regulated by the various components of the lipid sea in which they reside. Variations in lipid content clearly affect the activity of a variety of transport systems, and with advances in techniques for lipid analysis and a clearer vision of carrier structure, this area of research appears poised for major advances.

CLC Chloride Channels and Transporters: From Genes to Protein Structure, Pathology and Physiology

ABSTRACTCLC genes are expressed in species from bacteria to human and encode Cl−-channels or Cl−/H+-exchangers. CLC proteins assemble to dimers, with each monomer containing an ion translocation pathway. Some mammalian isoforms need essential β -subunits (barttin and Ostm1). Crystal structures of bacterial CLC Cl−/H+-exchangers, combined with transport analysis of mammalian and bacterial CLCs, yielded surprising insights into their structure and function. The large cytosolic carboxy-termini of eukaryotic CLCs contain CBS domains, which may modulate transport activity. Some of these have been crystallized.Mammals express nine CLC isoforms that differ in tissue distribution and subcellular localization. Some of these are plasma membrane Cl− channels, which play important roles in transepithelial transport and in dampening muscle excitability. Other CLC proteins localize mainly to the endosomal-lysosomal system where they may facilitate luminal acidification or regulate luminal chloride concentration. All vesicular CLCs may be Cl−/H+-exchangers, as shown for the endosomal ClC-4 and -5 proteins. Human diseases and knockout mouse models have yielded important insights into their physiology and pathology. Phenotypes and diseases include myotonia, renal salt wasting, kidney stones, deafness, blindness, male infertility, leukodystrophy, osteopetrosis, lysosomal storage disease and defective endocytosis, demonstrating the broad physiological role of CLC-mediated anion transport.

Roles for KCC Transporters in the Maintenance of Lens Transparency

To determine whether the potassium chloride cotransporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency. RT-PCR was performed on RNA extracted from fiber cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively. Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 microM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral fiber cells located up to 150 microm in from the lens capsule, whereas deeper fiber cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 microM) revealed that this damage was initiated primarily by the swelling of peripheral fiber cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 microm) of cell damage that was initiated by the dilation of the extracellular space between fiber cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral fiber cells but extensive cell swelling in deeper fiber cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane. The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral fiber cells in a dynamic fashion. These results indicate that, in addition to Cl- channels, KCC transporters play a role in mediating a circulating flux of Cl- ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens fiber cells.

Gαo2Regulates Vesicular Glutamate Transporter Activity by Changing Its Chloride Dependence

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

Granulocyte-Macrophage Colony-stimulating Factor Signals for Increased Glucose Transport via Phosphatidylinositol 3-Kinase- and Hydrogen Peroxide-dependent Mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent K(m) for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the alpha subunit of the GM-CSF receptor (alpha GMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity alpha GMR and in human cells expressing the high affinity alpha beta GMR complex. We identified a Src homology 3 domain-binding motif in alpha GMR at residues 358-361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to alpha GMR was obtained by co-immunoprecipitation with antibodies to alpha GMR and p85, and an alpha GMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of alpha GMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H(2)O(2)), generated by ligand or antibody binding to alpha GMR, as the initiating factor. Catalase treatment abrogated GM-CSF- or anti-alpha GMR antibody-stimulated glucose uptake in alpha GMR-expressing oocytes, and H(2)O(2) activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to alpha GMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H(2)O(2) generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated alpha subunit of the GM-CSF receptor.

Leptin transport across the blood–brain barrier of the Koletsky rat is not mediated by a product of the leptin receptor gene

Obesity in humans is thought to be caused by a resistance to leptin. Currently, the evidence suggests that this resistance is caused by an impaired transport of leptin across the blood–brain barrier (BBB). It has been assumed that the short form of the leptin receptor, which is a splice variant of the gene which produces all known leptin receptors, is the leptin transporter, but evidence for this is mixed. The Koletsky rat model should provide a clear answer as to whether transport is dependent on leptin receptors as it does not express any functional receptors. The transport of intravenous leptin across the BBB of the Koletsky rat has been found to be greatly reduced, but evidence for a residual of transport makes it unclear whether the transporter is essentially absent or simply saturated by the high levels of leptin in the serum. Here we used the brain perfusion method to negate the influence of serum levels. We found that, whereas no transport of intravenous leptin occurred in the obese Koletsky, the rate of transport was no different from controls when brain perfusion was used. Leptin was transported completely across the BBB, was saturable, and had the same distribution among brain regions as previously found in normal weight mice (highest transport into the hippocampus and hypothalamus, lowest in the frontal cortex). We conclude that a leptin transporter and possibly its gene have yet to be identified and that the short form likely plays a role in the modulation of transport activity.

The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions.

The NHE family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H(+) for extracellular Na(+). This review focuses on the only ubiquitously expressed isoform, NHE1, which is localized at the plasma membrane where it plays a critical role in intracellular pH (pHi) and cell volume homeostasis. All NHE isoforms share a similar topology: an N-terminus of 12 transmembrane (TM) alpha-helices that collectively function in ion exchange, and a C-terminal cytoplasmic regulatory domain that modulates transport activity by the TM domain. Extracellular signals, mediated by diverse classes of cell-surface receptors, regulate NHE1 activity through distinct signaling networks that converge to directly modify the C-terminal regulatory domain. Modifications in the C-terminus, including phosphorylation and the binding of regulatory proteins, control transport activity by altering the affinity of the TM domain for intracellular H(+). Recently, it was determined that NHE1 also functions as a membrane anchor for the actin-based cytoskeleton, independently of its role in ion translocation. Through its effects on pHi homeostasis, cell volume, and the actin cortical network, NHE1 regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation.