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Biomedical and Pharmaceutical Sciences Faculty Publications

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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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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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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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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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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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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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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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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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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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Effects Of Ecotropic Murine Retroviruses On The Dual-Function Cell Surface Receptor/Basic Amino Acid Transporter, Hao Wang, Esther Dechant, Michael Kavanaugh, R. Alan North, David Kabat Jan 1992

Effects Of Ecotropic Murine Retroviruses On The Dual-Function Cell Surface Receptor/Basic Amino Acid Transporter, Hao Wang, Esther Dechant, Michael Kavanaugh, R. Alan North, David Kabat

Biomedical and Pharmaceutical Sciences Faculty Publications

The widely expressed Na(+)-independent transporter for basic amino acids (system y+) is the cell surface receptor (ecoR) for ecotropic host-range mouse retroviruses (murine leukemia viruses (MuLVs)), a class of retroviruses that naturally infects only mice or rats. Accordingly, expression of mouse ecoR cDNA in mink CCL64 fibroblasts yields cells (CEN cells) that have y+ transporter activity above the endogenous background and that bind and are infected by ecotropic MuLVs. The effect of ecotropic MuLV infection on expression of y+ transporter was analyzed in mouse and in mink CEN fibroblasts. Chronic infection with ecotropic MuLVs caused 50-70% loss (down-modulation) of mouse …

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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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Cooperative Interactions Among Subunits Of A Voltage-Dependent Potassium Channel. Evidence From Expression Of Concatenated Cdnas, Raymond S. Hurst, Michael Kavanaugh, Jerrel Yakel, John P. Adelman, R. Alan North Jan 1992

Cooperative Interactions Among Subunits Of A Voltage-Dependent Potassium Channel. Evidence From Expression Of Concatenated Cdnas, Raymond S. Hurst, Michael Kavanaugh, Jerrel Yakel, John P. Adelman, R. Alan North

Biomedical and Pharmaceutical Sciences Faculty Publications

Four copies of the coding sequence for a voltage-dependent potassium channel (RBK1, rat Kv1.1) were ligated contiguously and transcribed in vitro. The resulting RNA encodes four covalently linked subunit domains ([4]RBK1). Injection of this RNA into Xenopus oocytes resulted in the expression of voltage-dependent potassium currents. A single amino acid substitution, Tyr-->Val, located within the outer mouth of the pore, introduced into the equivalent position of any of the four domains, reduced affinity for external tetraethylammonium by approximately the same amount. In constructs containing 0, 1, 2, 3, or 4 Tyr residues the free energy of binding tetraethylammonium was …

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Interaction Between Tetraethylammonium And Amino Acid Residues In The Pore Of Cloned Voltage-Dependent Potassium Channels, Michael Kavanaugh, Michael D. Varnum, Peregrine B. Osborne, Macdonald J. Christie, Andreas E. Busch, John P. Adelman, R. Alan North Jan 1991

Interaction Between Tetraethylammonium And Amino Acid Residues In The Pore Of Cloned Voltage-Dependent Potassium Channels, Michael Kavanaugh, Michael D. Varnum, Peregrine B. Osborne, Macdonald J. Christie, Andreas E. Busch, John P. Adelman, R. Alan North

Biomedical and Pharmaceutical Sciences Faculty Publications

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 …

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Structure And Function Of Human Hemoglobin Covalently Labeled With Periodate-Oxidized Adenosine Triphosphate, Michael Kavanaugh, Max F. Perutz, Giulio Fermi, Daniel T. Shih, Richard T. Jones Jan 1989

Structure And Function Of Human Hemoglobin Covalently Labeled With Periodate-Oxidized Adenosine Triphosphate, Michael Kavanaugh, Max F. Perutz, Giulio Fermi, Daniel T. Shih, Richard T. Jones

Biomedical and Pharmaceutical Sciences Faculty Publications

Periodate-oxidized adenosine triphosphate (o-ATP), a ribose ring-opened dialdehyde derivative of ATP, reacts specifically with human deoxyhemoglobin to give a single major covalently modified product after reduction with sodium borohydride. This product, designated di-ATP Hb, was isolated using ion-exchange chromatography and shown to have incorporated two molecules of o-ATP/tetramer. Peptide mapping and x-ray crystallography at 2.8-A resolution indicate that a covalent adduct is formed between the ligand and residues Lys-82 EF6 of each beta chain in the organic phosphate-binding site of the molecule. di-ATP Hb exhibits a significantly decreased oxygen affinity (P50 = 20.8 mm Hg versus 5.8 mm Hg control; …

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Identification Of The Binding Subunit Of The Sigma-Type Opiate Receptor By Photoaffinity Labeling With 1-(4-Azido-2-Methyl[6-3h]Phenyl)-3-(2-Methyl[4,6-3h]Phenyl)Guanidine, Michael Kavanaugh, Barbara C. Tester, Michael W. Scherz, John F. W. Keana, Eckard Weber Jan 1988

Identification Of The Binding Subunit Of The Sigma-Type Opiate Receptor By Photoaffinity Labeling With 1-(4-Azido-2-Methyl[6-3h]Phenyl)-3-(2-Methyl[4,6-3h]Phenyl)Guanidine, Michael Kavanaugh, Barbara C. Tester, Michael W. Scherz, John F. W. Keana, Eckard Weber

Biomedical and Pharmaceutical Sciences Faculty Publications

The sigma-type opiate receptor is a distinct binding site in the brain that may mediate some of the psychotomimetic effects caused by benzomorphan opiates and phencyclidine in humans. We have developed a synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify the binding protein(s) of the sigma receptor, we have now synthesized a radiolabeled azide derivative of DTG, 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)-guanidine ([3H]N3DTG). In guinea pig brain membrane binding assays conducted in the dark, [3H]N3DTG bound reversibly, selectively, and with high affinity (Kd = 10 nM) to sigma receptors. The drug specificity profile of reversible [3H]-N3DTG binding was identical to that …

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