title: Characterization of the first honeybee Ca²⁺ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation.
abstract: The honeybee is a model system to study learning and memory, and Ca(2+) signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca(2+) channel subunit. We identified two splice variants of the Apis CaVβ Ca(2+) 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 Ba(2+) 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 Ca(2+) 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 Ca(2+) channel activity by CaVβ subunit.
authors.affiliation: CRBM, UMR 5237, CNRS, Université de Montpellier I&II, Montpellier, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Cens
authors.forename: Matthieu
authors.initials: M
authors.lastname: Rousset
authors.forename: Claude
authors.initials: C
authors.lastname: Collet
authors.forename: Valérie
authors.initials: V
authors.lastname: Raymond
authors.forename: Fabien
authors.initials: F
authors.lastname: Démares
authors.forename: Annabelle
authors.initials: A
authors.lastname: Quintavalle
authors.forename: Michel
authors.initials: M
authors.lastname: Bellis
authors.forename: Yves
authors.initials: Y
authors.lastname: Le Conte
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chahine
authors.forename: Pierre
authors.initials: P
authors.lastname: Charnet
mesh_terms: D000200:Action Potentials; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005091:Exons; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D009474:Neurons / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D019476:Insect Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits