The neuronal microtubule-associated protein 1B is under homeoprotein transcriptional control.

To identify genes regulated by homeoprotein transcription factors in postnatal neurons, the DNA-binding domain (homeodomain) of Engrailed homeoprotein was internalized into rat cerebellum neurons. The internalized homeodomain (EnHD) acts as a competitive inhibitor of Engrailed and of several homeoproteins (Mainguy et al., 2000). Analysis by differential display revealed that microtubule-associated protein 1B (MAP1B) mRNA is upregulated by EnHD. This upregulation does not require protein synthesis, suggesting a direct effect of the homeodomain on MAP1B transcription. The promoter region of MAP1B was cut into several subdomains, and each subdomain was tested for its ability to bind Engrailed and EnHD and to associate with Engrailed-containing cerebellum nuclear extracts. In addition, the activity, and regulation by Engrailed, of each subdomain and of the entire promoter were evaluated in vivo by electroporation in the chick embryo neural tube. These experiments demonstrate that MAP1B promoter is regulated by Engrailed in vivo. Moreover, they show that one promoter domain that contains all ATTA homeoprotein cognate binding sites common to the rat and human genes is an essential element of this regulation. It is thus proposed that MAP1B, a cytoskeleton protein involved in neuronal growth and regeneration, is under homeoprotein transcriptional regulation.

Inhibition of the high-affinity brain glutamate transporter GLAST-1 via direct phosphorylation.

Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This downregulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.

Functional analysis of the high affinity, Na(+)-dependent glutamate transporter GLAST-1 by site-directed mutagenesis.

The reuptake of excitatory amino acids, such as glutamate, terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. The L-glutamate/L-aspartate transporter (GLAST-1) is the first member of the recently discovered glutamate transporter family, which includes GLT-1 and EAAC1. The neutral amino acid carrier ASCT1 is structurally closely related to this new family of membrane proteins. Transmembrane transport of neutral amino acids is expected to differ in its binding site from that of the acidic excitatory amino acids glutamate and aspartate. Three positively charged amino acid residues, Arg-122, Arg-280, Arg-479, and one polar Tyr-405 are conserved in all glutamate transporters. They are replaced by apolar amino acid residues in the ASCT1 sequence. We exchanged these residues in the GLAST-1-specific cDNA by site-directed mutagenesis. cRNAs of these mutants were expressed in the Xenopus oocyte system. The functional characterization of the mutants R122I and R280V and the double mutant R122I, R280V revealed that the mutations have no influence on the intrinsic properties and kinetics of glutamate transport but alter the Km-values for L-aspartate and the competitive inhibitor D,L-threo-3-hydroxy aspartate. Substitutions of Tyr-405 by Phe (Y405F) and Arg-479 (R479T) by Thr completely inactivate the glutamate transporter. Immunoprecipitations of [35S]methionine-labeled transporter molecules indicate similar expression levels of wild-type and mutant transporters. Immunostaining of oocyte sections clearly proves the correct targeting to and integration of the mutant GLAST-1 proteins in the plasma membrane. Our results suggest the pivotal function of the hydroxy group of the highly conserved Tyr-405 and the positively charged Arg-479 in the binding of the negatively charged acidic neurotransmitter glutamate.

Localization of N-glycosylation sites and functional role of the carbohydrate units of GLAST-1, a cloned rat brain L-glutamate/L-aspartate transporter.

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.

Functional properties and substrate specificity of the cloned L-glutamate/L-aspartate transporter GLAST-1 from rat brain expressed in Xenopus oocytes.

The rat brain L-glutamate/L-aspartate transporter GLAST-1 is a member of a family of Na(+)-dependent high-affinity L-glutamate transporters proposed to be involved in the termination and modulation of excitatory neurotransmitter signals. Application of electrophysiological and radiotracer techniques on Xenopus oocytes expressing cloned GLAST-1 revealed that the apparent Km value of the transporter for L-glutamate and Na+ ions did not depend on voltage while the maximal transport rate increased with more negative potentials, indicative of a low-field access channel. The apparent Km value of the transporter for L-glutamate depends on the Na+ concentration, suggesting that substrate and ions are transported by GLAST-1 in a simultaneous manner. All of the L-glutamate uptake blockers tested either were substrates or did not affect the current induced by L-glutamate. The changes in the amplitude of the current induced by simultaneous application of two substrates can be interpreted by a competition for one binding site.

Electrogenic L-glutamate uptake in Xenopus laevis oocytes expressing a cloned rat brain L-glutamate/L-aspartate transporter (GLAST-1).

The transport of L-glutamate into Xenopus laevis oocytes expressing the cloned L-glutamate/L-aspartate transporter (GLAST-1) from rat brain was studied using the voltage clamp technique. At a holding potential of -90 mV, a bath application of 100 microM L-glutamate induced an inward current (IGLAST) with an amplitude ranging from -5 to -30 nA. IGLAST did not require extracellular Ca2+, Mg2+, or Cl-, was larger at negative potentials, and did not reverse up to +80 mV. The current was dependent on external L-glutamate and Na+ with half-maximal amplitudes at 11 microM L-glutamate and 41 mM Na+. IGLAST saturated at 100 microM L-glutamate and 80 mM Na+. The Hill coefficient for Na+ and L-glutamate was 3.3 and 1.3, respectively, suggesting that 3 Na+ accompany the transport of 1 L-glutamate molecule. At low [Na+]o, IGLAST was enhanced by reducing [K+]o, an indication for the countertransport of K+. Reducing external pH from 7.4 to 6.0 did not change the amplitude of IGLAST. This argues against a glutamate/proton cotransport. The results provide evidence for GLAST-1 carrying out a high affinity, sodium-dependent L-glutamate transport with a proposed stoichiometry of 3 Na+, 1 L-glutamate-/1 K+.

Arterial baroreceptor function in differential cardiovascular adjustments induced by central thermal stimulation.

Dogs were anesthetized with sodium pentobarbital, relaxed with succinyl choline and were kept under artificial ventilation. Both carotid bifurcations were denervated and the Vagus nerves were cut in the neck. Regional blood flow in the skin and the intestine, cardiac output, heart rate and arterial pressure were determined before, during and after spinal cord heating and cooling. Further experiments were performed in which, in addition, sympathetic effects on the heart were excluded by exstirpation of the caudal cervical and stellate ganglia or by beta-receptor blockade. The cardiovascular responses were compared with those obtained in a preceding investigation from dogs with intact baroreceptors and vagus nerves. As in intact dogs, appropiate thermoregulatory adjustments of skin blood flow were induced by thermal stimulation of the spinal cord after baroreceptor denervation and vagotomy. However, blood pressure homeostasis was lost. The pattern of cardiovascular ajustments during heating consisted in cutaneous vasodilatation intestinal vasoconstriction and, due to sympathetic activation an increase of heart rate and cardiac output. This pattern was qualitatively identical with that intact animals. During spinal cord cooling the cardiovascular response pattern consisted in cutaneous vasoconstriction, intestinal vasoconstriction and, depending on cooling intensity, a reduced or unchanged sympathetic influence on the heart. This pattern differed considerably from what in intact animals but basic features were still present as indicated by opposite changes of cardiac and vascular sympathetic tone during cooling. It is concluded that the baroreceptor signals play no primary role in the generation of differential vasomotor responses under the present experimental conditions. This confirms assumptions made on the basis of observations in animals with intact baroreceptor input. However, baroreceptor signals contribute significantly to blood pressure homeostasis which is normally maintained during spinal thermal stimulation.