Résumé :
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The ANO5 gene is mutated in LGMD2L and a non-dysferlin Miyoshi myopathy, MMD3. Recessive ANO5 mutations are associated with sarcolemmal lesions and defective membrane repair. In European patients the ANO5 mutation, c.191dupA, is present in both MMD3 and LGMD2L patients and is emerging to be a common cause of adult onset muscular dystrophy. Previously dominant ANO5 mutations have been linked with a bone fragility disorder gnatho-diaphyseal dysplasia 1 (GDD1, MIM166260) where patients show no muscle involvement.ANO5 belongs to the anoctamin protein family, 10 human proteins (ANO 1-10) sharing a similar structure consisting of eight transmembrane domains, a re-entry loop domain and the novel DUF590 domain. Most anoctamins including ANO5 contain a nucleotide binding domain (cNMP domain) and a subset, including ANO5, have a C-terminal PDZ domain. Recently ANO1 and ANO2 have been shown to function as calcium activated chloride channels (CaCCs), which are ion channels gated by increases in intracellular Ca2+ concentration. Heterologous expression of ANO1 and ANO2 in various cell lines produced chloride currents that displayed biophysical and pharmacological properties typical of endogenous CaCCs identified in many different cell types and tissues. CaCC currents are important for diverse cellular functions-epithelial transport, cell volume regulation, olfactory and photoreceptor transduction, cardiac membrane excitability, and smooth muscle contraction. The ANO5 gene shows high expression in skeletal muscle and, although poorly characterized, it is likely to be membrane bound. Our hypothesis is that ANO5 encodes a CaCC in skeletal muscle. To investigate this we have generated monoclonal antibodies to ANO5 using specific N-terminal and C-terminal peptides. We are currently analyzing test hybridomas and have identified ANO5 antibody secreting clones, which detect a protein of the predicted size (107kDa) in muscle cells. Several of the hybridomas show staining in cultured myotubes. The hybridomas are being characterized further using C2C12 cells overexpressing ANO5 fusion proteins, muscle tissue and C2C12 ANO5 knockdown cells. These antibodies will be used for immunodiagnostics and research.
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