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

Abstract

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.