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Murviel-lès-Montpellier, France

Senatore A.,University of Waterloo | Monteil A.,French National Center for Scientific Research | Monteil A.,French Institute of Health and Medical Research | Monteil A.,Universites Montpellier I and | And 3 more authors.
PLoS ONE | Year: 2013

NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG+ impermeant and blockable with 10 μM Gd3+ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83. © 2013 Senatore et al. Source

Hippert C.,French National Center for Scientific Research | Hippert C.,Universites Montpellier I and | Ibanes S.,French National Center for Scientific Research | Ibanes S.,Universites Montpellier I and | And 9 more authors.
PLoS ONE | Year: 2012

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea. © 2012 Hippert et al. Source

Ayoub M.A.,Universites Montpellier I and | Ayoub M.A.,University of Western Australia | Trinquet E.,Cisbio Bioassays | Pfleger K.D.G.,University of Western Australia | Pin J.-P.,Universites Montpellier I and
FASEB Journal | Year: 2010

Although many G protein-coupled receptors (GPCRs) are known to activate multiple signaling pathways by coupling to different types of G proteins or by promoting G protein-independent events, how this occurs remains unclear. Using bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer, we provide evidence for protease-activated receptor 1 (PAR1) forming preassembled complexes with Gαi1 but not Gα12. PAR1 activation appears to rapidly induce transient Gαi1 activation (t1/2 = 4.13 s) but late and stable recruitment of Gα12 (t1/2 = 8.8 min) in parallel with β-arrestin 1 (t1/2 = 7.5 min). However, there is no significant difference in the potency of the agonist-dependent response between Gαi1, Gα12, and β-arrestin 1 (EC50 values 0.48, 0.30, and 0.15 nM, respectively). Although it seems β-arrestin 1 is recruited to preassembled PAR1-Gαi1 complexes, this appears unlikely with Gα12, suggesting 2 distinct receptor populations. Of note, we observed a different Gα12 association mode with other GPCRs, indicating that preassembly and association dynamics may be specific properties of a receptor-G protein pair. Furthermore, the Gα C terminus appears to play different roles in the distinct association modes. Consequently, G protein preassembly or recruitment may constitute novel mechanisms for controlling the kinetics and multitude of GPCR signaling pathways. © FASEB. Source

Weiss N.,University of Calgary | Weiss N.,French Institute of Health and Medical Research | Weiss N.,Joseph Fourier University | Hameed S.,University of Calgary | And 28 more authors.
Journal of Biological Chemistry | Year: 2012

T-type calcium channels represent a key pathway for Ca 2+entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca v3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca v3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca v3.2 channels in MPC9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca 2+ channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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