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Mar. Drugs 2014, 12(9), 4743-4755; doi:10.3390/md12094743

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

1
Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia
2
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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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. 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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