Spadin as a new antidepressant: absence of TREK-1-related side effects.

Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin.

To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C(2) domains of RIM display properties analogous to those of the C(2)B domain of synaptotagmin-I. Thus, RIM-C(2)A and RIM-C(2)B bind in a Ca(2+)-independent manner to alpha1B, the pore-forming subunit of N-type Ca(2+) channels (EC(50) = approximately 20 nm). They also weakly interact with the alpha1C but not the alpha1D subunit of L-type Ca(2+) channels. In addition, the C(2) domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nm, respectively, for RIM-C(2)A and 224 and 16 nm for RIM-C(2)B. The interactions of the C(2) domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca(2+). Thus, in the presence of Ca(2+) (EC(50) = approximately 75 microm) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C(2) domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.

Cysteine string proteins are associated with cortical granules of Xenopus laevis oocytes.

Cysteine string proteins (csps) are associated with secretory organelles in a wide range of eukaryotic cells. Functional studies of these proteins indicate that they subserve one or more vital steps in the pathway of regulated exocytosis. Here, we document the presence of csps in fully grown (stage VI) oocytes of the frog, Xenopus laevis. Both Northern and immunoblot data support the conclusion that csps are expressed in these cells. In addition, immunoreactive csp is seen even at the earliest stage of oocyte development, namely, in stage I oocytes. Finally, immunoblot and immunocytochemical results indicate that csps are associated with cortical granules of stage II-VI oocytes. These observations suggest that csps participate in the cortical reaction that underlies the sustained block to polyspermy in Xenopus eggs. Moreover, because of the relative ease of manipulating cells as large as Xenopus oocytes, this system harbors considerable promise as a model for studying the role of csps and other proteins in exocytotic events.

Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5.

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to alpha-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.

VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts.

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca(2+)-triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca(2+)-evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca(2+)-dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca(2+) concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion.

Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca(2+)-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic alpha-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca(2+)-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion. High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca(2+)-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.

Structure, functional expression, and cerebral localization of the levocabastine-sensitive neurotensin/neuromedin N receptor from mouse brain.

This work describes the cloning and expression of the levocabastine-sensitive neurotensin (NT) receptor from mouse brain. The receptor protein comprises 417 amino acids and bears the characteristics of G-protein-coupled receptors. This new NT receptor (NTR) type is 39% homologous to, but pharmacologically distinct from, the only other NTR cloned to date from the rat brain and the human HT29 cell line. When the receptor is expressed in Xenopus laevis oocytes, the H1 antihistaminic drug levocabastine, like NT and neuromedin N, triggers an inward current. The pharmacological properties of this receptor correspond to those of the low-affinity, levocabastine-sensitive NT binding site described initially in membranes prepared from rat and mouse brain. It is expressed maximally in the cerebellum, hippocampus, piriform cortex, and neocortex of adult mouse brain.

Widespread expression of human cysteine string proteins.

We present the nucleotide and deduced amino acid sequence of a human systeine string protein (csp) and a unique truncated csp variant that derives from the retention of an exonic sequence that introduces a premature, in-frame stop codon. Low stringency Southern analysis is compatible with the presence of a single human csp gene. Northern analysis reveals that human csp mRNA has both a more heterogeneous size distribution and a more widespread tissue distribution than previously reported for other csps. Exemplifying this is the fact that csp immunoreactivity is detected in Triton X-114 extracts of human blood, an observation which may facilitate blood-based diagnostic assays of csp status in man.

Expression cloning of a protective Leishmania antigen.

Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice. An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II. A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.

Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain.

MbIRK3, mbGIRK2 and mbGIRK3 K+ channels cDNAs have been cloned from adult mouse brain. These cDNAs encode polypeptides of 445, 414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane-spanning domains M1 and M2 and a pore-forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMK1 and GIRK1. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRK1 (60%) than to ROMK1 and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K(+)-selective currents very similar to IRK1 and GIRK1, respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G-protein coupled inward rectifier K+ channels which has been observed through their functional coupling with co-expressed delta-opioid receptors. These results provide the first evidence that the GIRK family, as the IRK family, is composed of multiple genes with members specifically expressed in the nervous system.

Molecular cloning of a murine N-type calcium channel alpha 1 subunit. Evidence for isoforms, brain distribution, and chromosomal localization.

A cDNA encoding a N-type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N-type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N-type Ca2+ channel gene has been localized on the chromosome 2, band A.

Characterization and partial purification from pheochromocytoma cells of an endogenous equivalent of scyllatoxin, a scorpion toxin which blocks small conductance Ca(2+)-activated K+ channels.

This work describes the partial purification of a heat-stable peptide which has the same properties as the scorpion toxin, scyllatoxin, a specific blocker of one class of Ca(2+)-activated K+ channels: (i) it competes with [125I]apamin for binding to the same site, (ii) like apamin and scyllatoxin, it blocks the after-potential hyperpolarization in skeletal muscle cells in culture, (iii) like apamin and scyllatoxin, it contracts guinea-pig taenia coli relaxed by epinephrine, (iv) it cross-reacts with antibodies raised against scyllatoxin but not with antibodies raised against apamin.

[Anesthesia, awareness and wakefulness]. / Anestesia, coscienza e vigilanza.

Drugs used in anesthesia do not always produce the expected neurophysiological results. By studying those cases in which the reappearance of intraoperative awareness is most common, an attempt was made to estimate how many and which levels of cerebral function could be identified. Four levels were recognised: awareness with or without memory, and wakefulness with or without dreams. These conditions are related to greater or lesser cortical activity. They can be identified using Tunstall's technique and by carefully interviewing the patient immediately after the operation.

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression.

A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization.

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.

Antibodies reveal the cytolocalization and subunit structure of the 1,4-dihydropyridine component of the neuronal Ca2+ channel.

Rabbit brain synaptosomes bind the 1,4-dihydropyridine derivative (+)[3H]-PN 200-110 with an equilibrium dissociation constant of 0.04 nM and a maximal binding capacity of 400 fmol/mg of protein. Using polyclonal antibodies raised against the different components of the skeletal muscle 1,4-dihydropyridine receptor, we have demonstrated that the brain and muscle receptors share the same subunit composition comprising a large polypeptide chain of Mr 140,000 associated by disulfide bridges with a smaller peptide of Mr 32,000. These antibodies have been used in immunofluorescence staining of brain sections. They reveal a distribution of the Ca2+ channel protein similar to that of 1,4-dihydropyridine binding sites with (+)[3H]PN 200-110 by the autoradiographic technique.