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Characterization of the first honeybee Ca2+ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation

  • Ion Channels, Receptors and Transporters
  • Published:
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Abstract

The honeybee is a model system to study learning and memory, and Ca2+ signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca2+ channel subunit. We identified two splice variants of the Apis CaVβ Ca2+ channel subunit (Am-CaVβ) and demonstrated expression in muscle and neurons. Although AmCaVβ shares with vertebrate CaVβ subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the CaV2 channels both, AmCaVβa and AmCaVβb, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate CaVβ2a subunit does. However, as opposed to CaVβ2a, slow inactivation induced by Am-CaVβa was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba2+ currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCaVβ variants may be important to couple cell signaling to Ca2+ entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCaVβa and AmCaVβb N termini, respectively, suggest that AmCaVβ splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca2+ channel activity by CaVβ subunit.

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Abbreviations

4-AP:

4-Amino-pyridine

BAPTA:

1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

DIV:

Days in vitro

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

NMDG:

N-Methyl-d-glucamine

PIP2:

Phosphoinositol-bi-phosphate

TEAOH:

Tetra-ethyl ammonium hydroxide

VGCC:

Voltage-gated Ca channels

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Acknowledgments

The authors would like to thank Menard, C., S. Machecourt, J.M. Donnay, J.C. Mazur, and M. Charreton for technical assistance; Dr. M. Morris for English proofreading; and J.P Vermandere, Jean Aptel (Experimental apiary from INRA Avignon), E. Carreras, D. Tavan (beekeepers at Saint Mathieu de Tréviers, France), and H. Rousset for their help.

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Authors and Affiliations

  1. CRBM, UMR 5237, CNRS, Université de Montpellier I&II, Montpellier, France, 34293

    Thierry Cens, Matthieu Rousset, Annabelle Quintavalle, Michel Bellis & Pierre Charnet

  2. UR406 Abeilles et Environnement, INRA, Avignon, France, 84914

    Claude Collet & Yves Le Conte

  3. Centre de Recherches sur la Cognition Animale, UMR 5169, CNRS, Université de Toulouse (UPS), Toulouse, France, 31062

    Valérie Raymond & Fabien Démares

  4. Centre de recherche, Institut universitaire en santé mentale de Québec, Québec, Canada, G1J 2G3

    Mohamed Chahine

  5. CRBM, UMR 5237, CNRS, Université de Montpellier II, 1919 route de Mende, Montpellier, 34293, France

    Pierre Charnet

Authors
  1. Thierry Cens
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  2. Matthieu Rousset
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  3. Claude Collet
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  4. Valérie Raymond
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Correspondence to Pierre Charnet.

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Thierry Cens and Matthieu Rousset contributed equally to this work.

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Cens, T., Rousset, M., Collet, C. et al. Characterization of the first honeybee Ca2+ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation. Pflugers Arch - Eur J Physiol 465, 985–996 (2013). https://doi.org/10.1007/s00424-013-1223-2

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  • DOI: https://doi.org/10.1007/s00424-013-1223-2

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