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Y Maulet

Publications and source records attributed to Y Maulet.

At least 19 recordsLinked to original sources
PubMed2001

Calcium-dependent translocation of synaptotagmin to the plasma membrane in the dendrites of developing neurones.

In neurones, the morphological complexity of the dendritic tree requires regulated growth and the appropriate targeting of membrane components. Accurate delivery of specific supplies depends on the translocation and fusion of transport vesicles. Vesicle SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and target membrane SNAREs play a central role in the correct execution of fusion events, and mediate interactions with molecules that endow the system with appropriate regulation. Synaptotagmins, a family of Ca(2+)-sensor proteins that includes neurone-specific members involved in regulating neurotransmitter exocytosis, are among the molecules that can tune the fusion mechanism. Using immunocytochemistry, confocal and electron microscopy, the localisation of synaptotagmin I in the dendrites of cultured rat hypothalamic neurones was demonstrated. Synaptotagmin labelling is concentrated at dendritic branch points, and in microprocesses. Following depolarisation, the N-terminal domain of synaptotagmin was detected at the extracellular surface of the dendritic plasma membrane. The insertion of synaptotagmin in the plasma membrane was elicited by L-type Ca(2+) channel activation and by mobilisation of the internal ryanodine-sensitive Ca(2+)stores. Furthermore, the localisation of L-type Ca(2+) channels and of ryanodine receptors, relative to the localisation of synaptotagmin in dendrites, suggests that both Ca(2+) entry and intracellular Ca(2+) stores may contribute to the fusion of dendritic transport vesicles with the membrane. Fusion is likely to involve SNAP-25 and syntaxin 1 as both proteins were also identified in dendrites. Taken together these results suggest a putative regulatory role of synaptotagmins in the membrane fusion events that contribute to shaping the dendritic tree during development.

Animals↗
PubMed2001

Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit.

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C). The interaction is restricted to the CB and IQ motifs involved in the calmodulin-mediated Ca(2+)-dependent inactivation of the DHPR, suggesting functional interactions between the two channels.

Amino Acid Sequence↗
PubMed2001

Interactions of calmodulin with two peptides derived from the c-terminal cytoplasmic domain of the Ca(v)1.2 Ca2+ channel provide evidence for a molecular switch involved in Ca2+-induced inactivation.

When opened by depolarization, L-type calcium channels are rapidly inactivated by the elevation of Ca(2+) concentration on the cytoplasmic side. Recent studies have shown that the interaction of calmodulin with the proximal part of the cytoplasmic C-terminal tail of the channel plays a prominent role in this modulation. Two motifs interacting with calmodulin in a Ca(2+)-dependent manner have been described: the IQ sequence and more recently the neighboring CB sequence. Here, using synthetic peptides and fusion proteins derived from the Ca(v)1.2 channel combined with biochemical techniques, we show that these two peptides are the only motifs of the cytoplasmic tail susceptible to interact with calmodulin. We determined the K(d) of the CB interaction with calmodulin to be 12 nm, i.e. below the K(d) of IQ-calmodulin, thereby precluding a competitive displacement of CB by IQ in the presence of Ca(2+). In place, we demonstrated that a ternary complex is formed at high Ca(2+) concentration, provided that calmodulin and the peptides are initially allowed to interact at a low Ca(2+) concentration. These results provide evidence that CB and IQ motifs interacting together with calmodulin constitute a minimal molecular switch leading to Ca(2+)-induced inactivation. In addition, we suggest that they could also be the tethering site of calmodulin.

Amino Acid Sequence↗
PubMed2001

Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain.

In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Ca(v)1.1) and RyR(1), whereas in the heart the signal is carried from the cardiac-type DHPR (Ca(v)1.2) to RyR(2) by calcium ions acting as an activator. Subtypes of both channels are expressed in the central nervous system, but their functions and mechanisms of coupling are still poorly understood. We show here that complexes immunoprecipitated from solubilized rat brain membranes with antibodies against DHPR of the Ca(v)1.2 or Ca(v)1.3 subtypes contain RyR. Only type-1 RyR is co-precipitated, although the major brain isoform is RyR(2). This suggests that, in neurons, DHPRs could communicate with RyRs by way of a strong molecular interaction and, more generally, that the physical link between DHPR and RyR shown to exist in skeletal muscle can be extended to other tissues.

Animals↗
PubMed1999

Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney.

ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K. The cDNA encodes a 77 kDa protein presenting 62% similarity with human ClC-Kb. The protein is monoglycosylated and is expressed primarily in the Xenopus kidney. It is localized in the basolateral membranes of proximal convoluted tubules of the nephron and in the apical region of the diluting segments. Heterologous expression of xClC-K in HEK-293 cells showed that the full-length protein is glycosylated and targeted to the cell membrane, but no associated Cl- current could be observed with the patch-clamp recording technique. N-glycosylation of both the native kidney channel and the recombinant protein expressed in HEK-293 conferred on them anomalous behaviour in denaturing PAGE, which is indicative of strong interactions at the extracellular side of the plasma membrane. The expression of ClC-K channels in both mesonephric and metanephric kidneys will permit further comparative physiological studies of Cl- permeabilities at the molecular level.

Amidohydrolases↗
PubMed1998

Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons.

Recently, two members of a new family of Ca2+ channel alpha1 subunits, alpha1G (or CavT.1) and alpha1H (or CavT.2), have been cloned and expressed. These alpha1 subunits generate Ba2+ currents similar to the T-type Ca2+ currents present in sensory neurons. Here, we use three methods to investigate whether the T currents of nodosus ganglion neurons are encoded by members of the CavT family. PCR detected the presence of mRNA encoding both alpha1G and alpha1H, as well as a third highly related sequence, alpha1I. In situ hybridizations performed on nodosus ganglia demonstrate a high expression of alpha1H subunit RNAs. Transfection of nodosus ganglion neurons with a generic antisense oligonucleotide against this new alpha1 subunit family selectively suppresses the low-voltage-activated Ca2+ current. The antisense oligonucleotide effect increased with time after transfection and reached a maximum 3 d after treatment, indicating a 2-3 d turnover for the alpha1 proteins. Taken together, these results suggest that the T-type current present in the sensory neurons is mainly attributable to alpha1H channels. In addition, taking advantage of the high specificity of the antisense ON to the cloned channels, we showed that T-type currents greatly slowed the repolarization occurring during an action potential and were responsible for up to 51% of the Ca2+ entry during spikes. Therefore, the antisense strategy clearly demonstrates the role of low-voltage-activated Ca2+ current in affecting the afterpotential properties and influencing the cell excitability. Such tools should be beneficial to further studies investigating physiological roles of T-type Ca2+ currents.

Action Potentials↗
PubMed1997

T-type Ca2+ current properties are not modified by Ca2+ channel beta subunit depletion in nodosus ganglion neurons.

At the molecular level, our knowledge of the low voltage-activated Ca2+ channel (T-type) has made little progress. Using an antisense strategy, we investigated the possibility that the T-type channels have a structure similar to high voltage-activated Ca2+ channels. It is assumed that high voltage-activated channels are made of at least three components: a pore forming alpha1 subunit combined with a cytoplasmic modulatory beta subunit and a primarily extracellular alpha2delta subunit. We have examined the effect of transfecting cranial primary sensory neurons with generic anti-beta antisense oligonucleotides. We show that in this cell type, blocking expression of all known beta gene products does not affect T-type current, although it greatly decreases the current amplitude of high voltage-activated channels and modifies their voltage dependence. This suggests that beta subunits are likely not constitutive of T-type Ca2+ channels in this cell type.

Calcium↗
PubMed1996

Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture: probing Ca2+ channel structure and function with antisense oligonucleotides.

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Animals↗
PubMed1994

Structural analysis of mouse glycine receptor alpha subunit genes. Identification and chromosomal localization of a novel variant.

The inhibitory glycine receptor is a ligand-gated ion channel protein that occurs in different developmentally regulated isoforms in the mammalian central nervous system. Here, we have analyzed genomic clones covering the coding regions of the murine glycine receptor alpha 1 and alpha 2 subunit genes. Both genes contain eight intronic regions with precisely conserved boundaries. The same structure was also found for seven exons of a third homologous gene, alpha 4, identified during screening. The predicted alpha 4 polypeptide displays very high homology to the alpha 2 subunit. Like the alpha 2 gene, the alpha 4 gene maps to the mouse X chromosome. Our data indicate that the genomic organization of glycine receptor alpha subunit genes is conserved during evolution.

Amino Acid Sequence↗
PubMed1994

Evolution of acetylcholinesterase transcripts and molecular forms during development in the central nervous system of the quail.

We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiple mRNA species (4.5 kb, 5.3 kb, and 6 kb); (b) translation and/or stability of the AChE protein; (c) production of active and inactive AChE molecules; (d) production of amphiphilic and nonamphiphilic AChE forms; and (e) proportions of tetrameric G4, dimeric G2, and monomeric G1 forms. The large transcripts present distinct temporal patterns and disappear in the adult, which possesses only the 4.5-kb mRNA; these changes are unlikely to be related to those observed for the AChE protein, because all transcripts seem to encode the same catalytic subunit (type T). In addition, the levels of mRNA and AChE are not correlated in the three regions, especially at the adult stage. The proportion of inactive AChE was found to be markedly higher at the hatching period (E16) than at earlier stages (E10 and E13) or in the adult. The G4 form is predominant already at E10, and in the adult its proportion reaches 80% of the activity in the cerebellum and optic lobes, and 65-70% in the neuroretina. This form is largely nonamphiphilic in embryonic tissues, but it becomes progressively more amphiphilic with development. Thus, the different processing and maturation steps appear to be regulated in an independent manner and potentially correspond to physiologically adaptative mechanisms.

Acetylcholinesterase↗
PubMed1992

Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein.

A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined. In brain, at least five different gephyrin mRNAs are generated by alternative splicing. Expression of gephyrin cDNAs in 293 kidney cells yields polypeptides reactive with a gephyrin-specific antibody, which coprecipitate with polymerized tubulin. Thus, gephyrin may define a novel type of microtubule-associated protein involved in membrane protein-cytoskeleton interactions.

Amino Acid Sequence↗
PubMed1991

Alternative splicing generates two isoforms of the alpha 2 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein which displays developmental heterogeneity in the mammalian central nervous system. Here we describe 2 novel cDNA variants of the rat GlyR alpha 2 subunit and demonstrate that alternative splicing generates these 2 isoforms. The deduced protein sequences (alpha 2A and alpha 2B) exhibit 99% identity with the previously characterized human alpha 2 subunit. In situ hybridization revealed expression of both alpha 2A and alpha 2B mRNAs in the prenatal rat brain, suggesting that these variant proteins may have a role in synaptogenesis. Heterologous expression in Xenopus oocytes showed that the more abundantly expressed alpha 2A subunit forms strychnine-sensitive ion channels which resemble human alpha 2 subunit GlyRs in their electrophysiological properties.

Amino Acid Sequence↗
PubMed1990

Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: alternative coding exons contain inverted repeat sequences.

Polymorphic forms of acetylcholinesterase are tethered extracellularly either as dimers membrane-anchored by a glycophospholipid or as catalytic subunits disulfidelinked to a collagen tail that associates with the basal lamina. Genomic clones of acetylcholinesterase from T. californica revealed that individual enzyme forms are encoded within a single gene that yields multiple mRNAs. Each enzyme form is encoded in three exons: the first two exons, bases -22 to 1502 and 1503 to 1669, encode sequence common to both forms, while alternative third exons encode a hydrophobic C-terminal region, to which a glycophospholipid is added upon processing, and a nonprocessed C-terminus, yielding a catalytic subunit that disulfide-links with a collagen-like structural unit. The 3' untranslated region of each alternative exon contains tandem repeat sequences that are inverted with respect to the other exon. This may either dictate alternative exon usage by formation of cis stem-loops or affect the abundance of translatable mRNA by trans-hybridization between the alternative spliced mRNA species.

Acetylcholinesterase↗
PubMed1989

Differences in structure and distribution of the molecular forms of acetylcholinesterase.

Two structurally distinct molecular forms of acetylcholinesterase are found in the electric organs of Torpedo californica. One form is dimensionally asymmetric and composed of heterologous subunits. The other form is hydrophobic and composed of homologous subunits. Sequence-specific antibodies were raised against a synthetic peptide corresponding to the COOH-terminal region (Lys560-Leu575) of the catalytic subunits of the asymmetric form of acetylcholinesterase. These antibodies reacted with the asymmetric form of acetylcholinesterase, but not with the hydrophobic form. These results confirm recent studies suggesting that the COOH-terminal domain of the asymmetric form differs from that of the hydrophobic form, and represent the first demonstration of antibodies selective for the catalytic subunits of the asymmetric form. In addition, the reactive epitope of a monoclonal antibody (4E7), previously shown to be selective for the hydrophobic form of acetylcholinesterase, has been identified as an N-linked complex carbohydrate, thus defining posttranslational differences between the two forms. These two form-selective antibodies, as well as panselective polyclonal and monoclonal antibodies, were used in light and electron microscopic immunolocalization studies to investigate the distribution of the two forms of acetylcholinesterase in the electric organ of Torpedo. Both forms were localized almost exclusively to the innervated surface of the electrocytes. However, they were differentially distributed along the innervated surface. Specific asymmetric-form immunoreactivity was restricted to areas of synaptic apposition and to the invaginations of the postsynaptic membrane that form the synaptic gutters. In contrast, immunoreactivity attributable to the hydrophobic form was selectively found along the non-synaptic surface of the nerve terminals and was not observed in the synaptic cleft or in the invaginations of the postsynaptic membrane. This differential distribution suggests that the two forms of acetylcholinesterase may play different roles in regulating the local concentration of acetylcholine in the synapse.

Acetylcholinesterase↗
PubMed1988

Multiple messenger RNA species give rise to the structural diversity in acetylcholinesterase.

Acetylcholinesterase exists predominantly as a secreted enzyme which remains cell-associated at specific extracellular locations. Its extensive structural diversity appears responsible for the unique cellular disposition of the enzyme. To examine the molecular basis of the structural divergence of acetylcholinesterase species, we hybridized total RNA from Torpedo californica electric organ with restriction fragments from a cDNA encoding the catalytic subunits of asymmetric species of acetylcholinesterase. Multiple RNA species up to 14 kilobases in length can be detected on Northern blots using a full-length cDNA for hybridization. Each of these RNA species also hybridizes with smaller restriction fragments within the open reading frame and 3'-untranslated region of the cDNA. This indicates that the entire open reading frame plus the 3'-untranslated region is contained in the large RNA species. RNase protection experiments revealed at least three points of divergence for the message species. One occurs within the COOH-terminal portion of the open reading frame at a position just 5' to the TGA stop codon. This divergence accounts for the two classes of acetylcholinesterase found in abundance in Torpedo. The site of splicing has been further defined by isolating a genomic clone containing the exon serving as the potential splice donor. We find a divergence between the cDNA and genomic DNA at the position estimated by the protection experiments. A less abundant divergence in mRNA can also be detected in the 3'-untranslated region. Another divergence occurs as a deleted sequence within the 5'-noncoding region and may be important for controlling translation efficiency. Since it is hypothesized that a single gene encodes acetylcholinesterase, the divergences in the very 3' region of the open reading frame and the 5'-noncoding region correspond to presumed splice junction boundaries where alternative RNA splicing occurs.

Acetylcholinesterase↗
PubMed1986

Primary structure of acetylcholinesterase: implications for regulation and function.

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.

Acetylcholinesterase↗
PubMed1984

Selective solubilization by melittin of glycophorin A and acetylcholinesterase from human erythrocyte ghosts.

Melittin, the main basic and hydrophobic peptide of bee venom, has been used for solubilizing membrane components of the human erythrocyte ghost. Up to 1.0 mM, it does not extract any phospholipid. Between 0.1 and 1.0 mM, it solubilizes partially glycophorin A and acetylcholinesterase. When the membrane is first degraded by phospholipase A2, the solubilization of both proteins by melittin is total, and 48% of the phospholipids are removed, mainly as lysoproducts, whereas phospholipase A2, by itself, has no solubilizing properties. In its melittin-solubilized state, acetylcholinesterase is in a dimeric form and displays a slow time-dependent irreversible inactivation. Triton X-100 at 1.0% (v/v) interrupts the inactivation. We suggest that melittin binds to the hydrophobic site of acetylcholinesterase which anchors it in the lipid bilayer.

Acetylcholinesterase↗