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

Marine Drugs

Ilya Pozdnyakov¹  Olga Matantseva¹  Yuri Negulyaev¹ 

[1] Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg 194064, Russia; E-Mails:

关键词: dinoflagellates;    ion channels;    patch-clamp;    spheroplasts;   

DOI  :  10.3390/md12094743

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.