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

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 tec...

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Autores principales: Pozdnyakov, Ilya, Matantseva, Olga, Negulyaev, Yuri, Skarlato, Sergei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178496/
https://www.ncbi.nlm.nih.gov/pubmed/25199048
http://dx.doi.org/10.3390/md12094743
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author Pozdnyakov, Ilya
Matantseva, Olga
Negulyaev, Yuri
Skarlato, Sergei
author_facet Pozdnyakov, Ilya
Matantseva, Olga
Negulyaev, Yuri
Skarlato, Sergei
author_sort Pozdnyakov, Ilya
collection PubMed
description 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.
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spelling pubmed-41784962014-10-02 Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping Pozdnyakov, Ilya Matantseva, Olga Negulyaev, Yuri Skarlato, Sergei Mar Drugs Article 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. MDPI 2014-09-05 /pmc/articles/PMC4178496/ /pubmed/25199048 http://dx.doi.org/10.3390/md12094743 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Pozdnyakov, Ilya
Matantseva, Olga
Negulyaev, Yuri
Skarlato, Sergei
Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping
title Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping
title_full Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping
title_fullStr Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping
title_full_unstemmed Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping
title_short Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping
title_sort obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178496/
https://www.ncbi.nlm.nih.gov/pubmed/25199048
http://dx.doi.org/10.3390/md12094743
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