Advertisement

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

  • Thierry Cens
  • Matthieu Rousset
  • Claude Collet
  • Valérie Raymond
  • Fabien Démares
  • Annabelle Quintavalle
  • Michel Bellis
  • Yves Le Conte
  • Mohamed Chahine
  • Pierre CharnetEmail author
Ion Channels, Receptors and Transporters

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.

Keywords

Apis mellifera Ca signaling PI3kinase Voltage-clamp 

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

Notes

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.

Supplementary material

424_2013_1223_MOESM1_ESM.pdf (671 kb)
ESM 1 (PDF 671 kb)

References

  1. 1.
    Arikkath J, Campbell KP (2003) Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr Opin Neurobiol 13:298–307PubMedCrossRefGoogle Scholar
  2. 2.
    Buraei Z, Yang J (2010) The beta subunit of voltage-gated Ca2+ channels. Physiol Rev 90:1461–1506PubMedCrossRefGoogle Scholar
  3. 3.
    Cain SM, Snutch TP (2011) Voltage-gated calcium channels and disease. Biofactors 37:197–205PubMedCrossRefGoogle Scholar
  4. 4.
    Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16:521–555PubMedCrossRefGoogle Scholar
  5. 5.
    Cens T, Restituito S, Rousset M, Charnet P (2005) Role of beta subunits in voltage-gated calcium channel function. In: Zamponi GW (ed) Eurekah.com and Kluwer Academics/Plenum Publishers, pp. 95–112Google Scholar
  6. 6.
    Chen YH, Li MH, Zhang Y, He LL, Yamada Y, Fitzmaurice A, Shen Y, Zhang H, Tong L, Yang J (2004) Structural basis of the alpha(1)-beta subunit interaction of voltage-gated Ca(2+) channels. Nature 429:675–680PubMedCrossRefGoogle Scholar
  7. 7.
    Chien AJ, Carr KM, Shirokov RE, Rios E, Hosey MM (1996) Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function. J Biol Chem 271:26465–26468PubMedCrossRefGoogle Scholar
  8. 8.
    Collet C (2009) Excitation-contraction coupling in skeletal muscle fibers from adult domestic honeybee. Pflugers Arch 458:601–612PubMedCrossRefGoogle Scholar
  9. 9.
    Collet C, Belzunces L (2007) Excitable properties of adult skeletal muscle fibres from the honeybee Apis mellifera. J Exp Biol 210:454–464PubMedCrossRefGoogle Scholar
  10. 10.
    Ebert AM, McAnelly CA, Srinivasan A, Linker JL, Horne WA, Garrity DM (2008) Ca2+ channel-independent requirement for MAGUK family CACNB4 genes in initiation of zebrafish epiboly. Proc Natl Acad Sci USA 105:198–203PubMedCrossRefGoogle Scholar
  11. 11.
    Gielow ML, Gu GG, Singh S (1995) Resolution and pharmacological analysis of the voltage-dependent calcium channels of Drosophila larval muscles. J Neurosci 15:6085–6093PubMedGoogle Scholar
  12. 12.
    Glanzman DL (2010) Common mechanisms of synaptic plasticity in vertebrates and invertebrates. Curr Biol 20:R31–R36PubMedCrossRefGoogle Scholar
  13. 13.
    Grunewald B (2003) Differential expression of voltage-sensitive K+ and Ca2+ currents in neurons of the honeybee olfactory pathway. J Exp Biol 206:117–129PubMedCrossRefGoogle Scholar
  14. 14.
    Grunewald B, Wersing A, Wustenberg DG (2004) Learning channels. Cellular physiology of odor processing neurons within the honeybee brain. Acta Biol Hung 55:53–63PubMedCrossRefGoogle Scholar
  15. 15.
    Hanlon MR, Berrow NS, Dolphin AC, Wallace BA (1999) Modelling of a voltage-dependent Ca2+ channel beta subunit as a basis for understanding its functional properties. FEBS Lett 445:366–370PubMedCrossRefGoogle Scholar
  16. 16.
    He LL, Zhang Y, Chen YH, Yamada Y, Yang J (2007) Functional modularity of the beta-subunit of voltage-gated Ca2+ channels. Biophys J 93:834–845PubMedCrossRefGoogle Scholar
  17. 17.
    Hendrich J, Van Minh AT, Heblich F, Nieto-Rostro M, Watschinger K, Striessnig J, Wratten J, Davies A, Dolphin AC (2008) Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin. Proc Natl Acad Sci U S A 105:3628–3633PubMedCrossRefGoogle Scholar
  18. 18.
    Husch A, Paehler M, Fusca D, Paeger L, Kloppenburg P (2009) Calcium current diversity in physiologically different local interneuron types of the antennal lobe. J Neurosci 29:716–726PubMedCrossRefGoogle Scholar
  19. 19.
    Iwamoto M, Oiki S (2012) Amphipathic antenna of an inward rectifier K+ channel responds to changes in the inner membrane leaflet. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1217323110
  20. 20.
    Kloppenburg P, Kirchhof BS, Mercer AR (1999) Voltage-activated currents from adult honeybee (Apis mellifera) antennal motor neurons recorded in vitro and in situ. J Neurophysiol 81:39–48PubMedGoogle Scholar
  21. 21.
    Kohn AB, Roberts-Misterly JM, Anderson PA, Greenberg RM (2003) Creation by mutagenesis of a mammalian Ca(2+) channel beta subunit that confers praziquantel sensitivity to a mammalian Ca(2+) channel. Int J Parasitol 33:1303–1308PubMedCrossRefGoogle Scholar
  22. 22.
    Laurent S, Masson C, Jakob I (2002) Whole-cell recording from honeybee olfactory receptor neurons: ionic currents, membrane excitability and odourant response in developing workerbee and drone. Eur J Neurosci 15:1139–1152PubMedCrossRefGoogle Scholar
  23. 23.
    Menzel R (2012) The honeybee as a model for understanding the basis of cognition. Nat Rev Neurosci 13:758–768PubMedCrossRefGoogle Scholar
  24. 24.
    Miranda-Laferte E, Schmidt S, Jara AC, Neely A, Hidalgo P (2012) A short polybasic segment between the two conserved domains of the beta2a-subunit modulates the rate of inactivation of R-type calcium channel. J Biol Chem 287:32588–32597PubMedCrossRefGoogle Scholar
  25. 25.
    Olcese R, Qin N, Schneider T, Neely A, Wei X, Stefani E, Birnbaumer L (1994) The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit’s effect on activation. Neuron 13:1433–1438PubMedCrossRefGoogle Scholar
  26. 26.
    Opatowsky Y, Chen CC, Campbell KP, Hirsch JA (2004) Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha1 interaction domain. Neuron 42:387–399PubMedCrossRefGoogle Scholar
  27. 27.
    Perisse E, Raymond-Delpech V, Neant I, Matsumoto Y, Leclerc C, Moreau M, Sandoz JC (2009) Early calcium increase triggers the formation of olfactory long-term memory in honeybees. BMC Biol 7:30PubMedCrossRefGoogle Scholar
  28. 28.
    Raingo J, Castiglioni AJ, Lipscombe D (2007) Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors. Nat Neurosci 10:285–292PubMedCrossRefGoogle Scholar
  29. 29.
    Restituito S, Cens T, Barrere C, Geib S, Galas S, De Waard M, Charnet P (2000) The β2a subunit is a molecular groom for the Ca2+ channel inactivation gate. J Neurosci 20:9046–9052PubMedGoogle Scholar
  30. 30.
    Salvador-Recatala V, Greenberg RM (2010) The N terminus of a Schistosome beta subunit regulates inactivation and current density of a Cav2 channel. J Biol Chem 285:35878–35888PubMedCrossRefGoogle Scholar
  31. 31.
    Sandoz JC (2011) Behavioral and neurophysiological study of olfactory perception and learning in honeybees. Front Syst Neurosci 5:98PubMedCrossRefGoogle Scholar
  32. 32.
    Schafer S, Rosenboom H, Menzel R (1994) Ionic currents of Kenyon cells from the mushroom body of the honeybee. J Neurosci 14:4600–4612PubMedGoogle Scholar
  33. 33.
    Soderlund DM (2011) Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances. Arch Toxicol 86:165–186PubMedCrossRefGoogle Scholar
  34. 34.
    Tadmouri A, Kiyonaka S, Barbado M, Rousset M, Fablet K, Sawamura S, Bahembera E, Pernet-Gallay K, Arnoult C, Miki T, Sadoul K, Gory-Faure S, Lambrecht C, Lesage F, Akiyama S, Khochbin S, Baulande S, Janssens V, Andrieux A, Dolmetsch R, Ronjat M, Mori Y, De Waard M (2012) Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy. EMBO J 31:3730–3744PubMedCrossRefGoogle Scholar
  35. 35.
    Van Petegem F, Clark KA, Chatelain FC, Minor DL (2004) Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature 429:671–675PubMedCrossRefGoogle Scholar
  36. 36.
    Wang XH, Connor M, Wilson D, Wilson HI, Nicholson GM, Smith R, Shaw D, Mackay JP, Alewood PF, Christie MJ, King GF (2001) Discovery and structure of a potent and highly specific blocker of insect calcium channels. J Biol Chem 276:40306–40312PubMedGoogle Scholar
  37. 37.
    Weinstock GM, Robinson GE, Gibbs RA et al (2006) Insights into social insects from the genome of the honeybee Apis mellifera. Nature 443:931–949CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Thierry Cens
    • 1
  • Matthieu Rousset
    • 1
  • Claude Collet
    • 2
  • Valérie Raymond
    • 3
  • Fabien Démares
    • 3
  • Annabelle Quintavalle
    • 1
  • Michel Bellis
    • 1
  • Yves Le Conte
    • 2
  • Mohamed Chahine
    • 4
  • Pierre Charnet
    • 1
    • 5
    Email author
  1. 1.CRBM, UMR 5237CNRS, Université de Montpellier I&IIMontpellierFrance
  2. 2.UR406 Abeilles et EnvironnementINRAAvignonFrance
  3. 3.Centre de Recherches sur la Cognition Animale, UMR 5169CNRS, Université de Toulouse (UPS)ToulouseFrance
  4. 4.Centre de rechercheInstitut universitaire en santé mentale de QuébecQuébecCanada
  5. 5.CRBM, UMR 5237 CNRS, Université de Montpellier IIMontpellierFrance

Personalised recommendations