Transport systems specific for L-gutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/Laspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxyaspartate. In situ hybridization reveals a ikingly high density of GLAST mRNA in the Purkiqje cell layer qf cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.In the mammalian central nervous system L-glutamate is the main transmitter for most excitatory neurons, which are involved in complex physiological processes, such as learning and memory (1). The excitatory signal is generally removed by reuptake of the amino acids into presynaptic terminals and surrounding glia cells by high-affinity transport systems, which appear to play an important role in the regulation of synaptic transmission (2). Conventional biochemical approaches have resulted in the partial purification of these proteins (3)(4)(5). To date, three distinct systems have been described based on different ion requirements: a Na+-dependent system (3-6), a chloride-dependent system (7, 8), and a Na+-and Cl--independent system that is stimulated by Ca2+ (9, 10).Here we report the isolation of a eukaryotic glutamate/ aspartate transporter (GLAST) from rat brain and its characterization at the cDNAt and protein level. The deduced primary structure shows appreciable similarity to bacterial glutamate and dicarboxylate transporters. Expression of GLAST in Xenopus oocytes demonstrates that it is a highaffinity, Na+-dependent L-glutamate/L-aspartate transporter. GLAST mRNA is exclusively expressed in brain; it is primarily localized in the cerebellar Purkinje cell layer and is less dense throughout the cerebrum.MATERIALS AND METHODS supplier's instructions. Immunoprecipitation was performed using C-GLAST-GST, an affinity-purified antibody raised against a recombinant fusion protein consisting of the 49 C-terminal amino acids of GLAST and glutathione S-transferase (pGEX-lN vector; Amrad, Victoria, Australia).Injection of GLAST cRNA into Xenopus Oocytes and Analysis of the Expressed Amino Acid Transport. Oocytes were isolated from Xenopus laevis as described (13), separated by gentle agitation in collagenase type 11 (2...

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-m microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry Ϸ10 6 strands complementary to one of the tags. About 10 5 copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue-or disease-directed, low-density planar microarrays.DNA analysis ͉ gene expression ͉ parallel cloning ͉ fluid microarray

The L-glutamate transporter GLAST-1 belongs to the newly discovered family of Na(+)-dependent, high-affinity glutamate transporters, which are involved in the regulation of synaptic excitatory neurotransmitter concentration in mammalian brain. The members of this family have a similar topological organisation with at least six transmembrane helices (TMHs) and two putative N-glycosylation sites located in the extracellular loop connecting TMH 3 and TMH 4. Besides these two conserved N-glycosylation motifs at Asn206 and Asn216, GLAST-1 possesses an additional one at Asn35. The putative N-glycosylation consensus motifs (Asn-Xaa-Ser/Thr) were deleted by replacement of Asn206 and/or Asn216 by Thr using site-directed mutagenesis (mutants N206T, N216T and N206,216T). The cDNAs encoding wild-type GLAST-1 and the three glycosylation-defective transport proteins were expressed in the Xenopus laevis oocyte system. Immunoprecipitation of the [35S]methionine-labeled and glycopeptidase-F-treated transporter molecules indicates that GLAST-1 is glycosylated at Asn206 and Asn216, whereas Asn35 remains unglycosylated. To assess a possible functional role of the two glycosylation sites wild-type and glycosylation-deficient GLAST-1 were expressed in Xenopus oocytes and characterized functionally by using the whole-cell voltage-clamp technique. The results prove that N-glycosylation has no impact on the transport activity of GLAST-1.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.