• Corpus ID: 11590051

Biophysical characterization of the honeybee DSC 1 orthologue reveals a novel voltage-dependent Ca 2 + channel subfamily : CaV 4 Pascal

@inproceedings{Badaroudine2016BiophysicalCO,
  title={Biophysical characterization of the honeybee DSC 1 orthologue reveals a novel voltage-dependent Ca 2 + channel subfamily : CaV 4 Pascal},
  author={Gosselin ‐ Badaroudine and Moreau and Simard and Cens and Rousset and Collet and Charnet and Chahine},
  year={2016}
}

• Gosselin ‐ Badaroudine, Moreau, +5 authors Chahine
• Published 2016
• Biology

Voltage-gated Na and Ca channels (NaV and CaV) belong to the large family of ion channels and feature four homologous domains, each containing six transmembrane (TM) segments. NaV channels initiate the action potentials of many excitable cells, thus regulating their electrical signals. CaV channels have been attributed more diverse roles, which is expected, because they would have emerged earlier in evolution and are permeable to Ca, a major second messenger. They are involved in pacemaker cell action potentials, muscle contraction, and the Ca-dependent exocytosis of vesicles, which regulates hormone and neurotransmitter secretion (Catterall et al., 2005b). NaV channels would have evolved from CaV channels (Hille, 2001). This possibility is supported by the existence of proteins such as the Drosophila melanogaster Na channel 1 (DSC1) and its orthologues. Indeed, those channels feature selectivity filter sequences that would be representative of an intermediate sequence between that of canonical NaV and CaV channels (Zhou et al., 2004; Liebeskind et al., 2011). DSC1 was first identified in Drosophila using probes corresponding to the eel Na channel (Salkoff et al., 1987). Like other NaV and CaV channels, DSC1 is a 24-TM protein divided into four homologous domains, each featuring a voltage-sensitive domain resulting from the assembly of the first four TMs (S1–S4). The S5–S6 TMs of each domain assemble to form the pore domain, which is responsible for ion permeation and selectivity. A highly conserved motif located at the aperture of the pore is the main contributor to the selectivity of the 24-TM channels and is composed of one amino acid from each domain located between the helixes of the reentrant loop between S5 and S6 (Heinemann et al., 1992; Catterall et al., 2005b). Usually, only negatively charged amino acids (E-E-D-D or E-E-E-E in domains DI–DII–DIII–DIV) form this selectivity filter in CaV channels, whereas neutral and positively charged amino acids are involved in Na selective channels (D-EK-A for NaV1 channels; Heinemann et al., 1992; Catterall et al., 2005a; Stephens et al., 2015). The selectivity filter sequence for most DSC1 homologues (D-E-E-A) appears to be a hybrid of the sequences found in NaV1 and CaV channels. Because DSC1 homologues and NaV1 channels would have evolved from a common ancestor, Bilaterian voltage-gated Na channels (NaV) evolved from voltage-gated Ca channels (CaV). The Drosophila melanogaster Na channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between CaV and NaV channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca-permeable Na channel belonging to the NaV2 family because of its sequence similarity with NaV channels. This is despite insect NaV2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca than Na. In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd and Zn. These characteristics are reminiscent of CaV channels. We also show a strong selectivity for Ca and Ba ions, marginal permeability to Li, and impermeability to Mg and Na ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as CaV4 rather than NaV2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee’s channel, namely slow activation and inactivation kinetics and strong selectivity for Ca ions. Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca channel subfamily: CaV4