title: Obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping.
abstract: Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100-250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1-5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1-10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia. pozdnyakov@cytspb.rssi.ru.
authors.forename: Ilya
authors.initials: I
authors.lastname: Pozdnyakov
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia. matantseva@cytspb.rssi.ru.
authors.forename: Olga
authors.initials: O
authors.lastname: Matantseva
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia. yurineg@mail.cytspb.rssi.ru.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Negulyaev
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia. s_skarlato@yahoo.com.
authors.forename: Sergei
authors.initials: S
authors.lastname: Skarlato
mesh_terms: D059038:Black Sea; D002452:Cell Count; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D004141:Dinoflagellida / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D018978:Molting / Q000187:drug effects; D018408:Patch-Clamp Techniques / Q000379:methods*; D013104:Spheroplasts / Q000737:chemistry*
chemical_list: D007473:Ion Channels; D002482:Cellulose