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.
Highlights
Ion channels are protein complexes, which control the flow of ions down their electrochemical gradient through the cell membranes
It has been shown that Ca2+ and Ca2+-dependent K+ and Na+ channels control the motile behavior of paramecia and Chlamydomonas sp. [3,4,5], and H+ channels are responsible for activation of bioluminescence in dinoflagellates [6,7] and involved in pH homeostasis of coccolithophores [8]
Calcofluor White M2R (CFW) showed a sufficient decrease in the cellulose content of ecdysed cells as compared to untreated cells (Figure 2a,b). We used this phenomenon in order to patch-clamp just ecdysed P. minimum cells, taking into account that they must be more accessible for a micropipette
Summary
Ion channels are protein complexes, which control the flow of ions down their electrochemical gradient through the cell membranes. Dinoflagellates are ecologically important organisms with unique physiological and biochemical features [9] Investigation of their ion channels represents one of the most attractive research areas, because these membrane proteins play an essential role in cell processes, such as cell signaling, proliferation and apoptosis, endo- and exocytosis, etc. Kwok et al in 2007 obtained spheroplasts of a dinoflagellate Crypthecodinium cohnii using polyethylene glycol and observed a decreased amount of cellulose in the treated cells [23] Their findings can hardly be effectively used in electrophysiological studies, because polyethylene glycol dramatically changes the properties of membranes, e.g., it induces their fusion, and, may affect ion channel activity. One involved the triggering of ecdysis by physical stress conditions (centrifugation), whereas the other employed chemical treatment with 2,6-dichlorobenzonitrile (DCB), a well-known inhibitor of cellulose synthesis [24]
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