Neurotransmitter transporters. A tale of two families.
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Amino Acid Sequence↗
Biomedical subjects
S G Amara
Publications and source records attributed to S G Amara.
Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.
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 ions and one chloride ion.
Animals↗
Molecular mechanisms of cell-specific and regulated expression of the calcitonin/alpha-CGRP and beta-CGRP genes.
The calcitonin/CGRP gene family utilizes several fundamental mechanisms for regulation of gene expression. The structural diversity of the family depends both on tissue-specific RNA processing and the presence of multiple, independently regulated genes. Our laboratory has been studying the structure and expression of the rat calcitonin/alpha-CGRP and beta-CGRP genes. We have studied the processing of transcripts from these genes by introducing a variety of mutated and hybrid genes into several cell lines to identify sequences critical for processing regulation. These mutant genes have ranged from point mutations to exchanges of entire splice sites, as well as chimeric constructs between the calcitonin/alpha-CGRP and beta-CGRP genes. The beta-CGRP gene provides a unique insight into the role of cis-acting sequences in tissue-specific splicing events. The rat beta-CGRP gene has an overall structure similar to that of the calcitonin/alpha-CGRP gene, but the former lacks an exon encoding a calcitonin-like hormone. Although the beta-CGRP gene contains splice junction sequences analogous to those utilized for alternative splicing in the calcitonin/alpha-CGRP gene, alternatively spliced products from regions within the beta-CGRP gene are not observed. Substitution of specific domains from the calcitonin/alpha-CGRP gene into the beta-gene can reconstitute some, but not all, aspects of alternative RNA processing. The results of transfection studies suggest that multiple regions within these genes contribute to alternative RNA splicing.
Amino Acid Sequence↗
Localization of calcitonin gene-related peptide in the rat and human pituitary gland using immunocytochemistry and in situ hybridization. CGRP expression during ontogeny and after endocrine manipulations.
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Adrenalectomy↗
Functional expression and CNS distribution of a beta-alanine-sensitive neuronal GABA transporter.
The synaptic action of gamma-aminobutyric acid (GABA) is terminated by high affinity, Na(+)-dependent transport processes in both neurons and glia. We have isolated a novel GABA transporter cDNA, GAT-B, which encodes a high affinity (Km = 2.3 microM), Na(+)- and Cl(-)-dependent GABA transport protein that is potently blocked by beta-alanine, a compound generally considered a selective inhibitor of glial transport. However, in situ hybridization studies indicate that GAT-B mRNA is expressed predominantly within neurons. These data indicate that the neuronal-glial distinction of GABA transporters based on inhibitor sensitivities must be reconsidered and suggest a greater diversity of GABA transporters than has been predicted by previous pharmacologic studies.
Amino Acid Sequence↗
Families of twelve transmembrane domain transporters.
Several functionally distinct families of transport proteins share the general structural motif of twelve transmembrane domains. The number of membrane proteins known to possess this common feature continues to expand with the cloning of transporters for various neurotransmitters, nucleosides, osmolytes and basic amino acids, in addition to the previously defined families of facilitative and sodium-driven sugar transporters.
Animals↗
Cloning and expression of a cocaine-sensitive rat dopamine transporter.
The action of dopamine and other monoamine neurotransmitters at synapses is terminated predominantly by high-affinity reuptake into presynaptic terminals by specific sodium-dependent neurotransmitter transport proteins. A complementary DNA encoding a rat dopamine transporter has been isolated that exhibits high sequence similarity with the previously cloned norepinephrine and gamma-aminobutyric acid transporters. Transient expression of the complementary DNA in HeLa cells confirms the cocaine sensitivity of this transporter.
Amino Acid Sequence↗
Vaccinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters.
The vaccinia/T7 transient expression system, which results in rapid, high-level expression of proteins encoded by plasmids bearing T7 promoters, provides a powerful strategy for the expression cloning of membrane transporters. To test the feasibility of this approach, we introduced the rabbit Na+/glucose transporter by liposome-mediated transfection into vaccinia infected HeLa cells and determined the characteristics and sensitivity of induced [14C]alpha-methyl D-glucopyranoside uptake. We observed a rapid (4-12 h) expression of saturable (Kt = 342 microM) [14C]alpha-methyl D-glucopyranoside uptake following transfection, with substrate and inhibitor sensitivities of the native carrier, including Na+ and temperature dependence and appropriate phloridzin sensitivity (KI = 9.1 microM). The time-dependent increase in alpha-methyl D-glucopyranoside uptake coincided with a decline in endogenous Na+/D-aspartate transport. Maximal levels of expression achieved were nearly 10-fold higher than that reported for transient expression of Na+/glucose transporters in the COS cell system. Rate and dilution estimates demonstrates a sensitivity of detection of single clones diluted several thousand fold by nonspecific plasmid DNA. A further 3-fold increase in transport sensitivity was achieved after transfection of plasmid constructs bearing additional 5'-T7 stem-loop and 3'-T7 termination signals. When cell lines with low endogenous transport were coupled with substrates of high specific activity, as with measurements of induced [3H]gamma-aminobutyric acid uptake, we were able to detect expression from transporter bearing plasmids diluted as much as 10,000-fold by non-specific plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter.
At most synapses, chemical signalling is terminated by a rapid reaccumulation of neurotransmitter into presynaptic terminals. Uptake systems for the biogenic amines are the initial site of action for therapeutic antidepressants and drugs such as cocaine and the amphetamines. We have isolated a complementary DNA clone encoding a human noradrenaline transporter. The cDNA sequence predicts a protein of 617 amino acids, with 12-13 highly hydrophobic regions compatible with membrane-spanning domains. Expression of the cDNA clone in transfected HeLa cells indicates that noradrenaline transport activity is sodium-dependent and sensitive to selective noradrenaline transport inhibitors. Transporter RNA is localized to the brainstem and the adrenal gland. The predicted protein sequence demonstrates significant amino-acid identity with the Na+/gamma-aminobutyric acid transporter, thus identifying a new gene family for neurotransmitter transporter proteins. Analysis of its structure and function may lead to structure-based drug design for the treatment of human depression and could help determine whether transporter abnormalities underlie affective disorders.
Adrenal Glands↗
Sodium-dependent neurotransmitter reuptake systems.
The recent cloning of the gamma-aminobutyric acid and norepinephrine Na(+)-dependent neurotransmitter transporters has led to new approaches for understanding the structure and function of these important synaptic proteins and provides a conceptual model in which to consider recent advances in the physiology, pharmacology and biochemistry of this gene family.
Animals↗
Distinct, developmentally regulated brain mRNAs direct the synthesis of neurotransmitter transporters.
The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.
Aging↗
Localization of immunoreactivity for calcitonin gene-related peptide in the rat anterior pituitary during ontogeny and gonadal steroid manipulations and detection of its messenger ribonucleic acid.
Localization of calcitonin gene-related peptide (CGRP) expression in the rat anterior pituitary and its changes during ontogeny and after gonadal steroid manipulations were studied by immunocytochemistry, RIA, and in situ hybridization. Colocalization studies and the combined use of immunocytochemistry and in situ hybridization revealed that CGRP immunoreactivity is localized mainly in gonadotropes and alpha- and beta-CGRP messenger RNAs were detected in CGRP-immunoreactive cells. Immunoreactivity for CGRP also was detected in nerve fibers and colocalized with substance P immunoreactivity. Cells immunoreactive to CGRP antiserum were first detected in fetal rats at gestational day 18, and the incidence considerably increased between postnatal days 5 and 14. CGRP immunoreactivity was low in control adults of both sexes and in pregnant and ovariectomized females but increased in lactating, estrogen-supplemented ovariectomized and high-dose estrogen-treated females, and in high-dose estrogen-treated and castrated males. Testosterone supplement suppressed the effect of castration on CGRP immunoreactivity in males. Quantities of extractable immunoreactive CGRP under conditions of estrogen manipulation corresponded well to the immunocytochemical findings (females: controls, 96.4 +/- 13.1 fmol/gland; ovariectomized, 107.6 +/- 19.2; high-dose estrogen-treated, 212 +/- 23.0; estrogen-supplemented ovariectomized, 680 +/- 42.1). The present study suggests that pituitary CGRP is synthesized and stored in gonadotropes, is modulated by gonadal steroids, and may have a functional link with gonadotropins.
Aging↗
A device to rapidly core out tissue culture plates for autoradiographic visualization of cells transfected with cDNA libraries.
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Animals↗
cDNA sequence of the rat U snRNP-associated protein N: description of a potential Sm epitope.
Anti-Sm antibodies from a patient with systemic lupus erythematosus (SLE) were used to isolate cDNA clones encoding the snRNP-associated protein N from a rat brain derived cDNA library. The predicted primary structure of the 240 amino acid protein has a proline rich carboxyl terminus and shares a region of sequence similarity with other snRNP polypeptides, A and B/B'. Anti-Sm sera recognize a beta-galactosidase fusion protein containing only the carboxyl-terminal 80 amino acids of N; antibodies eluted from this fusion protein also react with A, B/B' and N on immunoblots, suggesting that these proteins share an Sm epitope located within this segment. Polyclonal antibodies raised against a 23 amino acid synthetic peptide derived from this conserved region of N recognize A, N and B/B' on immunoblots and can immunoprecipitate the Sm class of U snRNAs. These results confirm that this sequence defines a potential Sm epitope. RNA blotting analyses demonstrate that a 1.6 kb mRNA expressed predominantly in brain encodes the N polypeptide in both rats and humans. At low stringency rat N cDNA also hybridizes to a 1.3 kb mRNA species which encodes B/B', suggesting that N is structurally related to, but distinct from B/B'. Although B/B' proteins are thought to be expressed in all human cells, only N and B, but not B', are observed on immunoblots of human brain proteins probed with anti-Sm sera. The apparent difference in the complement of proteins associated with snRNP particles in human brain versus elsewhere suggests a possible mechanism for the regulation of brain-specific mRNA splicing.
Amino Acid Sequence↗
Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat.
Calcitonin gene-related peptide (CGRP) mRNA was localised to neurones of the dorsal root ganglia and motoneurones of the ventral horn in man and rat. Presence of alpha- and beta-CGRP mRNA was confirmed by Northern blot analysis of rat tissues which showed alpha-CGRP was the predominant gene. The distribution of CGRP gene transcripts corresponded with neurones displaying CGRP immunoreactivity in the ganglia of both species and in the rat ventral horn. In man few motoneurones were immunoreactive despite many expressing CGRP mRNA. In situ hybridisation revealed not only sensory but also motor neurones are sites of CGRP manufacture. Thus in conjunction with other evidence the present study reinforces the proposed muscle trophic role for this peptide.
Aged↗
Differential expression of alpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat.
Expression of the calcitonin gene-related peptide, alpha-calcitonin gene-related peptide (CGRP), and the homologous beta-CGRP were compared in sensory and enteric nerves of the rat. Analysis of CGRP-like immunoreactivity by cation exchange chromatography and radioimmunoassay showed that in the dorsal root ganglia, dorsal spinal cord and in those peripheral tissues where CGRP-like immunoreactivity is primarily localized to sensory fibres, alpha-CGRP concentrations were three to six times greater than beta-CGRP concentrations. In the intestine, however, beta-CGRP concentrations were up to seven times greater than alpha-CGRP concentrations. Only beta-CGRP was detected in the intestines of capsaicin-treated rats. Northern blot and in situ hybridization to alpha-CGRP- and beta-CGRP-specific probes showed that while both alpha-CGRP and beta-CGRP messenger ribonucleic acids occurred in the dorsal root ganglia, only beta-CGRP messenger ribonucleic acid occurred in the intestine, where it was localized to enteric neurons. Receptor binding sites on membranes of rat heart and colon had approximately equal affinities for alpha-CGRP and beta-CGRP. The two peptides were equipotent in increasing the rate and force of atrial contractions but alpha-CGRP was slightly (2.6 times) more potent than beta-CGRP in relaxing colonic smooth muscle. Thus, both alpha-CGRP and beta-CGRP occur in the rat nervous system and are both biologically active. Sensory neurons and enteric neurons have been identified as populations which preferentially express alpha-CGRP and beta-CGRP, respectively.
Animals↗
Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N.
Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules [small nuclear ribonucleoprotein (snRNP) particles]. In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified Mr 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as "N," is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone (lambda rb91) from a rat brain phage lambda gt11 cDNA expression library. On RNA blots, the 450-base-pair cDNA insert of this clone hybridized to a 1600-nucleotide mRNA species with an identical tissue distribution to N, suggesting that lambda rb91 encodes at least part of N. A longer cDNA clone was obtained by rescreening the library with lambda rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.
Animals↗
Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes.
To permit a molecular characterization of neurotransmitter transporter proteins, we have studied uptake activities induced in Xenopus laevis oocytes after injection of adult rat forebrain, cerebellum, brainstem, and spinal cord poly(A)+ RNA. L-Glutamate uptake could be observed as early as 24 hr after injection, was linearly related to the quantity of mRNA injected, and could be induced after injection of as little as 1 ng of cerebellar mRNA. Transport of radiolabeled L-glutamate, gamma-aminobutyric acid, glycine, dopamine, serotonin, and choline could be measured in single microinjected oocytes with a regional profile consistent with the anatomical distribution of particular neurotransmitter synthesizing soma. Forebrain L-glutamate and dopamine uptake, as well as cerebellar L-glutamate transport, were found to be Na+-dependent. Cerebellar mRNA-induced L-glutamate transport was both time and temperature-dependent, was saturable by substrate, suggesting a single activity with an apparent transport Km of 14.2 microM and a Vmax of 15.2 pmol/hr per oocyte, and was sensitive to inhibitors of brain L-glutamate transport. Thus, the oocyte L-glutamate transport induced by injection of adult rat cerebellar mRNA appears essentially identical to the high-affinity, Na+-dependent L-glutamate uptake found in brain slices and nerve terminals. Experiments with size-fractionated cerebellar mRNA reveal single, comigrating peaks for cerebellar L-glutamate and gamma-aminobutyric acid transport, with peak activity obtained in fractions of approximately 2.7 kilobases, suggesting the presence of single or similarly sized mRNAs encoding each of these activities.
Animals↗