Cloning and functional expression of voltage-gated ion channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis.

Cnidocytes were dissociated from the tentacles of the Portuguese Man O'War Physalia physalis using heat treatment, and purified using density centrifugation. Visual observation confirmed that these cnidocytes contained a nucleus, a cnidocyst and an apical stereocilium, confirming that the cells were intact. A cnidocyte-specific amplified cDNA library was then prepared using RNA isolated from the cnidocytes, and screened for voltage-gated ion channel subunits using conventional molecular cloning techniques. A variety of channel proteins were identified and full-length sequence obtained for two of them, a Ca(2+) channel beta subunit (PpCa(V)beta) and a Shaker-like K(+) channel (PpK(V)1). The location of the transcripts was confirmed by RT-PCR of total RNA isolated from individually selected and rinsed cnidocytes. The functional properties of these two channel proteins were characterized electrophysiologically using heterologous expression. PpCa(V)beta modulates currents carried by both cnidarian and mammalian alpha(1) subunits although the specifics of the modulation differ. PpK(V)1 produces fast transient outward currents that have properties typical of other Shaker channels. The possible role of these channel proteins in the behavior of cnidocytes is discussed.

Specific sites in the Beta Interaction Domain of a schistosome Ca2+ channel beta subunit are key to its role in sensitivity to the anti-schistosomal drug praziquantel.

Praziquantel, the drug of choice against schistosomiasis, disrupts calcium (Ca2+) homeostasis in schistosomes via an unknown mechanism. Voltage-gated Ca2+ channels are heteromultimeric transmembrane protein complexes that contribute to impulse propagation and also regulate intracellular Ca2+ levels. Beta subunits modulate the properties of the pore-forming alpha1 subunit of high voltage-activated Ca2+ channels. Unlike other Ca2+ channel beta subunits, which have current stimulatory effects, a beta subunit subtype found in S. mansoni (SmbetaA) and S. japonicum (Sjbeta) dramatically reduces current levels when co-expressed with Ca2+ channel alpha1 subunits in Xenopus oocytes. It also confers praziquantel sensitivity to the mammalian Cav2.3 alpha1 subunit. The Beta Interaction Domains (BIDs) of SmbetaA and Sjbeta lack 2 conserved serines that each constitute a consensus site for protein kinase C (PKC) phosphorylation. Here, we use site-directed mutagenesis of schistosome beta subunits to show that these unique functional properties are correlated with the absence of these consensus PKC sites in the BID. Furthermore, a second schistosome beta subunit subtype contains both serines in the BID, enhances currents through alpha1 subunits, and does not confer praziquantel sensitivity. Thus, phosphorylation sites in the BID may play important roles in defining the modulatory properties and pharmacological sensitivities of schistosome Ca2+ channel beta subunits.

Taurine-like Immunoreactivity in the Motor Nerve Net of the Jellyfish Cyanea capillata.

Two antisera against the sulfonated amino acid taurine were applied to subumbrella tissue of the jellyfish Cyanea capillata. Taurine-immunoreactive nerve nets were found in both the ectoderm and endoderm. The ectoderm had two morphologically and immunocytochemically distinct populations of neurons, the motor nerve net (MNN), which was immunoreactive to the taurine-like molecule, and the diffuse nerve net (DNN), which was immunoreactive to the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide). In the endoderm, immunoreactivity was found in the endodermal DNN. This localization was confirmed by double-labeling experiments, which also revealed that the endodermal DNN neurons may contain both taurine and FMRFamide-related peptide. The presence of a taurine immunoreactivity in the MNN supports the hypothesis that taurine or some chemically related compound is the neurotransmitter at synapses within the MNN of Cyanea.