Obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping

Mar Drugs. 2014 Sep 5;12(9):4743-55. doi: 10.3390/md12094743.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Black Sea
  • Cell Count
  • Cellulose / chemistry
  • Cellulose / metabolism
  • Dinoflagellida / chemistry*
  • Ion Channels / chemistry*
  • Molting / drug effects
  • Patch-Clamp Techniques / methods*
  • Spheroplasts / chemistry*

Substances

  • Ion Channels
  • Cellulose