Characterization of the first honeybee Ca2+ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation
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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.
KeywordsApis mellifera Ca signaling PI3kinase Voltage-clamp
Days in vitro
Tetra-ethyl ammonium hydroxide
Voltage-gated Ca channels
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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