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Mar. Drugs 2014, 12(9), 4743-4755;

Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping

Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia
Department of Medical Physics, St. Petersburg State Polytechnical University, Polytechnicheskaya ul. 29, St. Petersburg 195251, Russia
Author to whom correspondence should be addressed.
Received: 1 July 2014 / Revised: 18 August 2014 / Accepted: 26 August 2014 / Published: 5 September 2014
(This article belongs to the Special Issue Marine Dinoflagellates)
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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. View Full-Text
Keywords: dinoflagellates; ion channels; patch-clamp; spheroplasts dinoflagellates; ion channels; patch-clamp; spheroplasts

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Pozdnyakov, I.; Matantseva, O.; Negulyaev, Y.; Skarlato, S. Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping. Mar. Drugs 2014, 12, 4743-4755.

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