Kinetic properties of the channels formed by the bacillus thuringiensis insecticidal crystal protein Cry1C in the plasma membrane of Sf9 cells.
Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K(+) and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K(+). These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K(+) efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K(+) efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line.
Guihard
G
G
Biotechnology Research Institute, National Research Council, 6100 Royalmount Ave., Montreal, Quebec, H4P 2R2, Canada.
Vachon
V
V
Laprade
R
R
Schwartz
J L
JL
D000818:Animals; D001413:Bacillus thuringiensis / Q000187:drug effects*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins; D002462:Cell Membrane / Q000187:drug effects; D004731:Endotoxins / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000494:pharmacology*; D007473:Ion Channels / Q000187:drug effects; D007811:Lanthanum / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D010539:Permeability / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D013696:Temperature
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D007306:Insecticides; D007473:Ion Channels; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D007811:Lanthanum; D011188:Potassium