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Articles 61 - 90 of 95

Full-Text Articles in Pharmacy and Pharmaceutical Sciences

Dynamic Equilibrium Between Coupled And Uncoupled Modes Of A Neuronal Glutamate Transporter, Lars Borre, Michael Kavanaugh, Baruch I. Kanner Jan 2002

Dynamic Equilibrium Between Coupled And Uncoupled Modes Of A Neuronal Glutamate Transporter, Lars Borre, Michael Kavanaugh, Baruch I. Kanner

Biomedical and Pharmaceutical Sciences Faculty Publications

In the brain, the neurotransmitter glutamate is removed from the synaptic cleft by (Na+ + K+)-coupled transporters by an electrogenic process. Moreover, these transporters mediate a sodium- and glutamate-dependent uncoupled chloride conductance. In contrast to the wild type, the uptake of radiolabeled substrate by the I421C mutant is inhibited by the membrane-impermeant [2-(trimethylammonium)ethyl]methanethiosulfonate and also by other sulfhydryl reagents. In the wild-type and the unmodified mutant, substrate-induced currents are inwardly rectifying and reflect the sum of the coupled electrogenic flux and the anion conductance. Remarkably, the I421C mutant modified by sulfhydryl reagents exhibits currents that are non-rectifying …

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Truncated Forms Of The Dual Function Human Asct2 Neutral Amino Acid Transporter/Retroviral Receptor Are Translationally Initiated At Multiple Alternative Cug And Gug Codons, Chetankumar S. Tailor, Mariana Marin, Ali Nouri, Michael Kavanaugh, David Kabat Jan 2001

Truncated Forms Of The Dual Function Human Asct2 Neutral Amino Acid Transporter/Retroviral Receptor Are Translationally Initiated At Multiple Alternative Cug And Gug Codons, Chetankumar S. Tailor, Mariana Marin, Ali Nouri, Michael Kavanaugh, David Kabat

Biomedical and Pharmaceutical Sciences Faculty Publications

The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3′ noncoding region and a 627-bp 5′ truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino …

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Isolation Of Current Components And Partial Reaction Cycles In The Glial Glutamate Transporter Eaat2, Thomas S. Otis, Michael Kavanaugh Jan 2000

Isolation Of Current Components And Partial Reaction Cycles In The Glial Glutamate Transporter Eaat2, Thomas S. Otis, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of l-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN−, pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. …

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Pentameric Assembly Of A Neuronal Glutamate Transporter, Sepehr Eskandari, Michael Kreman, Michael Kavanaugh, Ernest M. Wright, Guido A. Zampighi Jan 2000

Pentameric Assembly Of A Neuronal Glutamate Transporter, Sepehr Eskandari, Michael Kreman, Michael Kavanaugh, Ernest M. Wright, Guido A. Zampighi

Biomedical and Pharmaceutical Sciences Faculty Publications

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number …

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Arginine 447 Plays A Pivotal Role In Substrate Interactions In A Neuronal Glutamate Transporter, Annie Bendahan, Ayelet Armon, Navid Madani, Michael Kavanaugh, Baruch I. Kanner Jan 2000

Arginine 447 Plays A Pivotal Role In Substrate Interactions In A Neuronal Glutamate Transporter, Annie Bendahan, Ayelet Armon, Navid Madani, Michael Kavanaugh, Baruch I. Kanner

Biomedical and Pharmaceutical Sciences Faculty Publications

Glutamate transporters from the central nervous system play a crucial role in the clearance of the transmitter from the synaptic cleft. Glutamate is cotransported with sodium ions, and the electrogenic translocation cycle is completed by countertransport of potassium. Mutants that cannot interact with potassium are only capable of catalyzing electroneutral exchange. Here we identify a residue involved in controlling substrate recognition in the neuronal transporter EAAC-1 that transports acidic amino acids as well as cysteine. When arginine 447, a residue conserved in all glutamate transporters, is replaced by cysteine, transport of glutamate or aspartate is abolished, but sodium-dependent cysteine transport …

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A Critical Site In The Core Of The Ccr5 Chemokine Receptor Required For Binding And Infectivity Of Human Immunodeficiency Virus Type 1, Salvatore J. Siciliano, Shawn E. Kuhmann, Youmin Weng, Navid Madani, Martin S. Springer, Janet E. Lineberger, Renee Danzeisen, Michael D. Miller, Michael Kavanaugh, Julie A. Demartino, David Kabat Jan 1999

A Critical Site In The Core Of The Ccr5 Chemokine Receptor Required For Binding And Infectivity Of Human Immunodeficiency Virus Type 1, Salvatore J. Siciliano, Shawn E. Kuhmann, Youmin Weng, Navid Madani, Martin S. Springer, Janet E. Lineberger, Renee Danzeisen, Michael D. Miller, Michael Kavanaugh, Julie A. Demartino, David Kabat

Biomedical and Pharmaceutical Sciences Faculty Publications

Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (∼98–99% identical) CCR5 of African green monkeys (AGMs) avidly binds β-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely …

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Neurotransmitter Transport: Models In Flux, Michael Kavanaugh Jan 1998

Neurotransmitter Transport: Models In Flux, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

Physiologists say that ions and neutral solutes can cross biological membranes via “transporters” and “channels.” We tend to think about the difference between transporters and channels in terms of gating mechanisms. Ion channels exhibit a wide range of selectivity properties and permeation rates, but their gating at the most basic level can be thought of in terms of a single barrier or gate acting as a switch. When the gate is closed, ions can’t permeate; when the gate is opened, a permeation pathway for ions allows flux, often at very high rates (up to 108/sec). Transporters and ion …

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Excitatory Amino Acid Transporters Of The Salamander Retina: Identification, Localization, And Function, Scott Eliasof, Jeffrey L. Arriza, Barbara H. Leighton, Michael Kavanaugh, Susan G. Amara Jan 1998

Excitatory Amino Acid Transporters Of The Salamander Retina: Identification, Localization, And Function, Scott Eliasof, Jeffrey L. Arriza, Barbara H. Leighton, Michael Kavanaugh, Susan G. Amara

Biomedical and Pharmaceutical Sciences Faculty Publications

The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed …

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Molecular Determinant Of Ion Selectivity Of A (Na+ + K+)-Coupled Rat Brain Glutamate Transporter, Yumin Zhang, Annie Bendahan, Ruth Zarbiv, Michael Kavanaugh, Baruch I. Kanner Jan 1998

Molecular Determinant Of Ion Selectivity Of A (Na+ + K+)-Coupled Rat Brain Glutamate Transporter, Yumin Zhang, Annie Bendahan, Ruth Zarbiv, Michael Kavanaugh, Baruch I. Kanner

Biomedical and Pharmaceutical Sciences Faculty Publications

Glutamate transporters remove this neurotransmitter from the synaptic cleft by a two-stage electrogenic process, in which glutamate is first cotransported with three sodium ions and a proton. Subsequently, the cycle is completed by translocation of a potassium ion in the opposite direction. Recently, we have identified an amino acid residue of the glutamate transporter GLT-1 (Glu-404) that influences potassium coupling. We have now analyzed the effect of seven other amino acid residues in the highly conserved region surrounding this site. One of these residues, Tyr-403, also proved important for potassium coupling, because mutation to Phe (Y403F) resulted in an electroneutral …

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Cysteine Scanning Of The Surroundings Of An Alkali-Ion Binding Site Of The Glutamate Transporter Glt-1 Reveals A Conformationally Sensitive Residue, Ruth Zarbiv, Myriam Grunewald, Michael Kavanaugh, Baruch I. Kanner Jan 1998

Cysteine Scanning Of The Surroundings Of An Alkali-Ion Binding Site Of The Glutamate Transporter Glt-1 Reveals A Conformationally Sensitive Residue, Ruth Zarbiv, Myriam Grunewald, Michael Kavanaugh, Baruch I. Kanner

Biomedical and Pharmaceutical Sciences Faculty Publications

Glutamate transporters remove this transmitter from the extracellular space by cotransport with three sodium ions and a proton. The cycle is completed by translocation of a potassium ion in the opposite direction. Recently we have identified two adjacent amino acid residues of the glutamate transporter GLT-1 that influence potassium coupling. Using the scanning cysteine accessibility method we have now explored the highly conserved region surrounding them. Replacement of each of the five consecutive residues 396–400 by cysteine abolished transport activity but at several other positions the substitution is tolerated. One residue, tyrosine 403, was identified where cysteine substitution renders the …

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Gp120 Envelope Glycoproteins Of Human Immunodeficiency Viruses Competitively Antagonize Signaling By Coreceptors Cxcr4 And Ccr5, Navid Madani, Susan L. Kozak, Michael Kavanaugh, David Kabat Jan 1998

Gp120 Envelope Glycoproteins Of Human Immunodeficiency Viruses Competitively Antagonize Signaling By Coreceptors Cxcr4 And Ccr5, Navid Madani, Susan L. Kozak, Michael Kavanaugh, David Kabat

Biomedical and Pharmaceutical Sciences Faculty Publications

Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K+ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1α for CXCR4 and MIP-1α; MIP-1β and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5–7 min and recovery time constants of 12–19 min. CXCR4 and CCR5 exhibited heterologous …

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Arachidonic Acid Activates A Proton Current In The Rat Glutamate Transporter Eaat4, Anastassios V. Tzingounis, Chien-Liang Lin, Jeffrey D. Rothstein, Michael Kavanaugh Jan 1998

Arachidonic Acid Activates A Proton Current In The Rat Glutamate Transporter Eaat4, Anastassios V. Tzingounis, Chien-Liang Lin, Jeffrey D. Rothstein, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate …

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Macroscopic And Microscopic Properties Of A Cloned Glutamate Transporter/Chloride Channel, Jacques I. Wadiche, Michael Kavanaugh Jan 1998

Macroscopic And Microscopic Properties Of A Cloned Glutamate Transporter/Chloride Channel, Jacques I. Wadiche, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

The behavior of a Cl− channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of ∼5Å. Glutamate flux exhibited a much greater temperature dependence than Cl− flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl− conductance and a maximum channelPo ≪ 1. The glutamate binding rate was similar to estimates …

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Multiple Ionic Conductances Of The Human Dopamine Transporter: The Actions Of Dopamine And Psychostimulants, Mark S. Sonders, Si-Jia Zhu, Nancy R. Zahniser, Michael Kavanaugh, Susan G. Amara Jan 1997

Multiple Ionic Conductances Of The Human Dopamine Transporter: The Actions Of Dopamine And Psychostimulants, Mark S. Sonders, Si-Jia Zhu, Nancy R. Zahniser, Michael Kavanaugh, Susan G. Amara

Biomedical and Pharmaceutical Sciences Faculty Publications

Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate “transport-associated” current. (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport-associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater …

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Excitatory Amino Acid Transporter 5, A Retinal Glutamate Transporter Coupled To A Chloride Conductance, Jeffrey L. Arriza, Scott Eliasof, Michael Kavanaugh, Susan G. Amara Jan 1997

Excitatory Amino Acid Transporter 5, A Retinal Glutamate Transporter Coupled To A Chloride Conductance, Jeffrey L. Arriza, Scott Eliasof, Michael Kavanaugh, Susan G. Amara

Biomedical and Pharmaceutical Sciences Faculty Publications

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-d-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel …

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Aspartate 19 And Glutamate 121 Are Critical For Transport Function Of The Myo-Inositol/H+ Symporter From Leishmania Donovani, Andreas Seyfang, Michael Kavanaugh, Scott M. Landfear Jan 1997

Aspartate 19 And Glutamate 121 Are Critical For Transport Function Of The Myo-Inositol/H+ Symporter From Leishmania Donovani, Andreas Seyfang, Michael Kavanaugh, Scott M. Landfear

Biomedical and Pharmaceutical Sciences Faculty Publications

The protozoan flagellate Leishmania donovani has an active myo-inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopusoocytes and overexpression in Leishmania transfectants to investigate the significance of acidic transmembrane residues for proton relay and inositol transport. MIT has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type …

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Tyrosine 140 Of The Γ-Aminobutyric Acid Transporter Gat-1 Plays A Critical Role In Neurotransmitter Recognition, Yona Bismuth, Michael Kavanaugh, Baruch I. Kanner Jan 1997

Tyrosine 140 Of The Γ-Aminobutyric Acid Transporter Gat-1 Plays A Critical Role In Neurotransmitter Recognition, Yona Bismuth, Michael Kavanaugh, Baruch I. Kanner

Biomedical and Pharmaceutical Sciences Faculty Publications

The γ-aminobutyric acid (GABA) transporter GAT-1 is located in nerve terminals and catalyzes the electrogenic reuptake of the neurotransmitter with two sodium ions and one chloride. We now identify a single tyrosine residue that is critical for GABA recognition and transport. It is completely conserved throughout the superfamily, and even substitution to the other aromatic amino acids, phenylalanine (Y140F) and tryptophan (Y140W), results in completely inactive transporters. Electrophysiological characterization reveals that both mutant transporters exhibit the sodium-dependent transient currents associated with sodium binding as well as the chloride-dependent lithium leak currents characteristic of GAT-1. On the other hand, in both …

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Mutation Of An Amino Acid Residue Influencing Potassium Coupling In The Glutamate Transporter Glt-1 Induces Obligate Exchange, Michael Kavanaugh, Annie Bendahan, Noa Zerangue, Yumin Zhang, Baruch I. Kanner Jan 1997

Mutation Of An Amino Acid Residue Influencing Potassium Coupling In The Glutamate Transporter Glt-1 Induces Obligate Exchange, Michael Kavanaugh, Annie Bendahan, Noa Zerangue, Yumin Zhang, Baruch I. Kanner

Biomedical and Pharmaceutical Sciences Faculty Publications

Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. After cotransport of glutamic acid with Na+, the cycle is completed by countertransport of K+. We have identified an amino acid residue (glutamate 404) influencing ion coupling in a domain of the transporter implicated previously in kainate binding. Mutation of this residue to aspartate (E404D) prevents both forward and reverse transport induced by K+. Sodium-dependent transmitter exchange and a transporter-mediated chloride conductance are unaffected by the mutation, indicating that this residue selectively influences potassium flux coupling. The results …

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Asct-1 Is A Neutral Amino Acid Exchanger With Chloride Channel Activity, Noa Zerangue, Michael Kavanaugh Jan 1996

Asct-1 Is A Neutral Amino Acid Exchanger With Chloride Channel Activity, Noa Zerangue, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

The ubiquitous transport activity known as system ASC is characterized by a preference for small neutral amino acids including alanine, serine, and cysteine. ASCT-1 and ASCT-2, recently cloned transporters exhibiting system ASC-like selectivity, are members of a major amino acid transporter family that includes a number of glutamate transporters. Here we show that ASCT1 functions as an electroneutral exchanger that mediates negligible net amino acid flux. The electrical currents previously shown to be associated with ASCT1-mediated transport result from activation of a thermodynamically uncoupled chloride conductance with permeation properties similar to those described for the glutamate transporter subfamily. Like glutamate …

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Kinetics And Stoichiometry Of A Proton/Myo-Inositol Cotransporter, Elizabeth M. Klamo, Mark E. Drew, Scott M. Landfear, Michael Kavanaugh Jan 1996

Kinetics And Stoichiometry Of A Proton/Myo-Inositol Cotransporter, Elizabeth M. Klamo, Mark E. Drew, Scott M. Landfear, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

Voltage clamp recording was used to measure steady-state and presteady-state currents mediated by a myo-inositol transporter cloned from Leishmania donovani and expressed in Xenopus oocytes. Application of myo-inositol resulted in inward currents, which did not require external sodium and which were increased by increasing the extracellular proton concentration and by membrane hyperpolarization. Alkalinization of the extracellular space occurred concomitantly with myo-inositol influx. Correlation of membrane currents with radiolabeled myo-inositol flux revealed that one positive charge is translocated with each molecule of myo-inositol, consistent with cotransport of one proton. The transport concentration dependence on both species …

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Differential Modulation Of Human Glutamate Transporter Subtypes By Arachidonic Acid, Noa Zerangue, Jeffrey L. Arriza, Susan G. Amara, Michael Kavanaugh Jan 1995

Differential Modulation Of Human Glutamate Transporter Subtypes By Arachidonic Acid, Noa Zerangue, Jeffrey L. Arriza, Susan G. Amara, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

Arachidonic acid has been proposed to be a messenger molecule released following synaptic activation of glutamate receptors and during ischemia. Here we demonstrate that micromolar levels of arachidonic acid inhibit glutamate uptake mediated by EAAT1, a human excitatory amino acid transporter widely expressed in brain and cerebellum, by reducing the maximal transport rate approximately 30%. In contrast, arachidonic acid increased transport mediated by EAAT2, a subtype abundantly expressed in forebrain and midbrain, by causing the apparent affinity for glutamate to increase more than 2-fold. The results demonstrate that the response of different glutamate transporter subtypes to arachidonic acid could influence …

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A Family Of Putative Receptor-Adenylate Cyclases From Leishmania Donovani, Marco A. Sanchez, David Zeoli, Elizabeth M. Klamo, Michael Kavanaugh, Scott M. Landfear Jan 1995

A Family Of Putative Receptor-Adenylate Cyclases From Leishmania Donovani, Marco A. Sanchez, David Zeoli, Elizabeth M. Klamo, Michael Kavanaugh, Scott M. Landfear

Biomedical and Pharmaceutical Sciences Faculty Publications

Leishmania parasites are exposed to pronounced changes in their environment during their life cycle as they migrate from the sandfly midgut to the insect proboscis and then into the phagolysosomes of the vertebrate macrophages. The developmental transformations that produce each life cycle stage of the parasite may be signaled in part by binding of environmental ligands to receptors which mediate transduction of extracellular signals. We have identified a family of five clustered genes in Leishmania donovani which may encode signal transduction receptors. The coding regions of two of these genes, designated rac-A and rac-B, have been sequenced and shown …

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Constitutive Ion Fluxes And Substrate Binding Domains Of Human Glutamate Transporters, Robert J. Vandenberg, Jeffrey L. Arriza, Susan G. Amara, Michael Kavanaugh Jan 1995

Constitutive Ion Fluxes And Substrate Binding Domains Of Human Glutamate Transporters, Robert J. Vandenberg, Jeffrey L. Arriza, Susan G. Amara, Michael Kavanaugh

Biomedical and Pharmaceutical Sciences Faculty Publications

Application of L-glutamate activates ionic currents in voltage-clamped Xenopus oocytes expressing cloned human excitatory amino acid transporters (EAATs). However, even in the absence of L-glutamate, the membrane conductance of oocytes expressing EAAT1 was significantly increased relative to oocytes expressing EAAT2 or control oocytes. Whereas transport mediated by EAAT2 is blocked by the non-transported competitive glutamate analog kainate (K = 14 μM), EAAT1 is relatively insensitive (K > 3 mM). Substitution of a block of 76 residues from EAAT2 into EAAT1, in which 18 residues varied from EAAT1, conferred high affinity kainate binding to EAAT1, and application of kainate to …

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Cloning Of The Cellular Receptor For Amphotropic Murine Retroviruses Reveals Homology To That For Gibbon Ape Leukemia Virus, Daniel G. Miller, Robert H. Edwards, A. Dusty Miller Jan 1994

Cloning Of The Cellular Receptor For Amphotropic Murine Retroviruses Reveals Homology To That For Gibbon Ape Leukemia Virus, Daniel G. Miller, Robert H. Edwards, A. Dusty Miller

Biomedical and Pharmaceutical Sciences Faculty Publications

The host and tissue specificity of retrovirus infection is largely determined by specific cellular receptors that mediate virus entry. Genes encoding these receptors are widely distributed in the genome, and the receptors identified to date show no sequence similarity. We have identified the cellular receptor for amphotropic murine retroviruses, Ram-1, by screening a rat cDNA expression library introduced into amphotropic virus-resistant hamster cells. The 656-amino acid receptor is homologous to the gibbon ape leukemia virus receptor at both hydrophobic termini but is highly divergent in the central hydrophilic region. Both receptors appear to be integral membrane proteins having multiple membrane-spanning …

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Control Of Cationic Amino Acid Transport And Retroviral Receptor Functions In A Membrane Protein Family, Michael Kavanaugh, Hao Wang, Zheng Zhang, Weibin Zhang, Yan-Na Wu, Esther Dechant, R. Alan North, David Kabat Jan 1994

Control Of Cationic Amino Acid Transport And Retroviral Receptor Functions In A Membrane Protein Family, Michael Kavanaugh, Hao Wang, Zheng Zhang, Weibin Zhang, Yan-Na Wu, Esther Dechant, R. Alan North, David Kabat

Biomedical and Pharmaceutical Sciences Faculty Publications

A partial cDNA sequence indicated that the T lymphocyte early-activation gene (Tea) encodes a protein related to the dual-function ecotropic retrovirus receptor/cationic amino acid transporter (ecoR/CAT1), and RNA blots suggested highest Tea expression in T lymphocytes and liver (MacLeod, C.L., Finley, K., Kakuda, D. Kozad, C.A., and Wilkinson, M.F. (1990) Mol. Cell. Biol. 7, 3663-3674). The sequence of full-length Tea cDNA from liver (3683 bases) predicts a 657-amino-acid protein (CAT2 alpha) with 12-14 transmembrane domains. A long (515 base) region with six initiation codons and termination codons precedes the translation start codon. The liver Tea cDNA is identical to Tea …

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Functional Expression Of Two Glucose Transporter Isoforms From The Parasitic Protozoan Leishmania Enriettii, Chris K. Langford, Brian M. Little, Michael Kavanaugh, Scott M. Landfear Jan 1994

Functional Expression Of Two Glucose Transporter Isoforms From The Parasitic Protozoan Leishmania Enriettii, Chris K. Langford, Brian M. Little, Michael Kavanaugh, Scott M. Landfear

Biomedical and Pharmaceutical Sciences Faculty Publications

The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport …

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Cell-Surface Receptors For Gibbon Ape Leukemia Virus And Amphotropic Murine Retrovirus Are Inducible Sodium-Dependent Phosphate Symporters, Michael Kavanaugh, Daniel G. Miller, Weibin Zhang, Wendy Law, Susan L. Kozak, David Kabat, A. Dusty Miller Jan 1994

Cell-Surface Receptors For Gibbon Ape Leukemia Virus And Amphotropic Murine Retrovirus Are Inducible Sodium-Dependent Phosphate Symporters, Michael Kavanaugh, Daniel G. Miller, Weibin Zhang, Wendy Law, Susan L. Kozak, David Kabat, A. Dusty Miller

Biomedical and Pharmaceutical Sciences Faculty Publications

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic retrovirus (Ram-1) are distinct but related proteins having multiple membrane-spanning regions. Distant homology with a putative phosphate permease of Neurospora crassa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mammalian cells, we have identified Glvr-1 and Ram-1 as sodium-dependent phosphate symporters. Two-electrode voltage-clamp analysis indicates net cation influx, suggesting that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by > 50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-1, indicating that Ram-1 is a major …

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Functional Comparisons Of Three Glutamate Transporter Subtypes Cloned From Human Motor Cortex, Jeffrey L. Arriza, Wendy A. Fairman, Jacques I. Wadiche, Geoffrey H. Murdoch, Michael Kavanaugh, Susan G. Amara Jan 1994

Functional Comparisons Of Three Glutamate Transporter Subtypes Cloned From Human Motor Cortex, Jeffrey L. Arriza, Wendy A. Fairman, Jacques I. Wadiche, Geoffrey H. Murdoch, Michael Kavanaugh, Susan G. Amara

Biomedical and Pharmaceutical Sciences Faculty Publications

Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non- nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each …

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Cloning And Expression Of A Human Neutral Amino Acid Transporter With Structural Similarity To The Glutamate Transporter Gene Family, Jeffrey L. Arriza, Michael Kavanaugh, Wendy A. Fairman, Yan-Na Wu, Geoffrey H. Murdoch, R. Alan North, Susan G. Amara Jan 1993

Cloning And Expression Of A Human Neutral Amino Acid Transporter With Structural Similarity To The Glutamate Transporter Gene Family, Jeffrey L. Arriza, Michael Kavanaugh, Wendy A. Fairman, Yan-Na Wu, Geoffrey H. Murdoch, R. Alan North, Susan G. Amara

Biomedical and Pharmaceutical Sciences Faculty Publications

A cDNA was isolated from human brain that encodes an amino acid sequence 34-39% identical to previously published glutamate transporter sequences. Injection of RNA transcribed from this cDNA into Xenopus oocytes resulted in expression of a transport activity with the properties of the neutral amino acid uptake system ASC. Superfusion of alanine, serine, and cysteine evoked sodium-dependent inward currents in voltage-clamped oocytes expressing the transporter. These currents were dose-dependent, stereospecific, and saturable, with Km values ranging from 29 to 88 microM. Northern blot analyses revealed ubiquitous expression of this gene, termed ASCT1, consistent with the general metabolic role ascribed to …

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Electrogenic Uptake Of Gamma-Aminobutyric Acid By A Cloned Transporter Expressed In Xenopus Oocytes, Michael Kavanaugh, Jeffrey L. Arriza, R. Alan North, Susan G. Amara Jan 1992

Electrogenic Uptake Of Gamma-Aminobutyric Acid By A Cloned Transporter Expressed In Xenopus Oocytes, Michael Kavanaugh, Jeffrey L. Arriza, R. Alan North, Susan G. Amara

Biomedical and Pharmaceutical Sciences Faculty Publications

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium …

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