0235 : In vivo overexpression of a cardiac sodium channel mutant in mice

Loss-of-function mutations in the cardiac Na+ channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced, in HEK cells, the dominant-negative effect of the R104W BrS mutation in Nav1.5, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein. To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAV).We used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. One-week old mice were injected with two AAV serotypes capsid 9: one, packaging the cardiac specific troponin-T promoter, the 5’ half of hSCN5A, the 5’ donor site of a synthetic intron and a highly recombinogenic sequence; and another, packaging the same recombinogenic sequence, the 3’ acceptor site of the synthetic intron, the 3’ half of hSCN5A, the gfp gene as a reporter, and the SV40 polyA signal. Six weeks after injection, the hSCN5A full-gene expression and the percentage of transduced cardiac cells were assessed by qPCR, western blot (WB) analysis and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patchclamp technique in isolated cardiomyocytes.Both WT and mutant human Nav1.5 transcripts and proteins were observed by RT-qPCR, WB and immunohistochemistry on injected-mice heart tissues. Patch-clamp recordings in WT-channel injected mice evidenced a two-fold increase of the Na+ current. In contrast, the cardiac Na+ current of R104Winjected mice was impaired (i.e. the current density was decreased by 45% and the activation was shifted by -4mV).Our data suggest that the trans-splicing and viral DNA recombination strategy using AAV9 serotype and a cardiac-specific promoter is successful to overexpress WT or mutant Na+ channels in mouse hearts. This approach allowed us to modulate the cardiac Na+ current in adult mice

Human genetic polymorphisms in T1R1 and T1R3 taste receptor subunits affect their function.

International audienceUmami is the typical taste induced by monosodium glutamate (MSG), which is thought to be detected by the heterodimeric G protein-coupled receptor, T1R1 and T1R3. Previously, we showed that MSG detection thresholds differ substantially between individuals and we further showed that nontaster and hypotaster subjects are associated with nonsynonymous single polymorphisms occurring in the T1R1 and T1R3 genes. Here, we show using functional expression that both amino acid substitutions (A110V and R507Q) in the N-terminal ligand-binding domain of T1R1 and the 2 other ones (F749S and R757C), located in the transmembrane domain of T1R3, severely impair in vitro T1R1/T1R3 response to MSG. A molecular model of the ligand-binding region of T1R1/T1R3 provides a mechanistic explanation supporting functional expression data. The data presented here support causal relations between the genotype and previous in vivo psychophysical studies in human evaluating sensitivity to MSG

Impact d’un environnement hyperosmolaire évalué sur les cellules épithéliales intestinales Caco-2

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Novel Putative Nicotinic Acetylcholine Receptor Subunit Genes, D α 5 , D α 6 and D α 7 , in Drosophila melanogaster Identify a New and Highly Conserved Target of Adenosine Deaminase Acting on RNA-Mediated A-to-I Pre-mRNA Editing

International audienceAbstract Genome analysis of the fruit fly Drosophila melanogaster reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in α-type nicotinic acetylcholine receptor subunits. The subunits are designated Dα5, Dα6, and Dα7. Cloning of the Dα5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dα6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dα6 involves exons encoding nAChR functional domains. The Dα6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dα6, four of which are also edited in the Dα6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to our understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels

The nicotinic acetylcholine receptor gene family of the malaria mosquito, Anopheles gambiae

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Sequence comparison of ACE1 the gene encoding acetylcholinesterase of class A in the two nematodes Caenorhabditis elegans and Caenorhabditis briggsea

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Molecular Cloning and Characterization of a cDNA Encoding AChE from Optic Lobe of Loligo Opalescences

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Expression of a single dimeric membrane-bound acetylcholinesterase in Parascaris equorum

International audienceA single form of cholinesterase was detected in the parasitic nematode Parascaris equorum and purified from a low-salt Triton X-100 extract of whole animals by affinity chromatography on an edrophonium–Sepharose matrix. Based on gel-filtration chromatography, sedimentation analysis and SDS–PAGE, such a cholinesterase is an amphiphilic globular (G 2 ) dimer (125–129 kDa, 6·1 S). It includes some hydrophobic domain other than phosphatidylinositol, which gives auto-aggregation, detergent interaction and also anchors the molecule to the cell membrane. The enzyme, probably functional in cholinergic neurotransmission, is an acetylcholinesterase showing a fairly low substrate specificity with thiocholine esters. Electrostatic interactions seem to play a major role in the catalytic activity. Studies with inhibitors gave complete inhibition with 1 m M eserine, low sensitivity for procainamide and for tetra(monoisopropyl)pyrophosphortetramide as well as higher inhibition with edrophonium chloride and 1,5- bis (4allyldimethylammoniumphenyl)-pentan-3-one dibromide. The enzyme also showed excess-substrate inhibition with acetylthiocholine. No cross-hybridization occurred between the gene(s) encoding acetylcholinesterase in P. equorum and ace -1 from the free-living nematode Caenorhabditis elegans . The expression of a single cholinesterase form in P. equorum , unusual in free-living nematodes, could be due to parasitic life adaptation with resulting reduction of locomotor activity

Molecular Polymorphism of Acetylcholinesterase in Hirudo medicinalis

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Acetylcholinesterase from Octopus vulgaris (Cephalopoda)

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