Tracking sodium channels in live cells: confocal imaging using fluorescently labeled toxins.
One particularly important class of ion channels in excitable cells are the voltage-dependent sodium channels (VDSC). Knowledge of the distribution of VDSC in living cells is important for studies of neuronal excitability, development, and plasticity. Here, we demonstrate a new method for visualizing the spatial distribution of VDSC in living cells. To illustrate the capabilities of the technique, the distribution of VDSC in GH3 cells was revealed with fluorescent derivatives of the alpha-type and beta-type scorpion toxins in conjunction with laser scanning confocal microscopy. Cells exhibited fluorescent hot spots on the surface of the membrane. This characteristic staining pattern was prevented by pre-incubation with unlabeled native toxins and blocked by membrane depolarization for alpha-type toxins. Labeling was not observed in cells lacking sodium channels (HEK 293) after incubation with fluorescent-labeled toxins. Image processing techniques were applied to identify the location of each cluster of labeled VDSC in these cells. The proposed method eliminates artefacts commonly introduced during sample preparation for immunostaining and should prove to be a valuable research tool for investigating VDSC distribution in living specimens.
Massensini
Andre R
AR
Brain Image Analysis Unit, Institute of Psychiatry, King's College London, London, UK
Suckling
John
J
Brammer
Michael J
MJ
Moraes-Santos
Tasso
T
Gomez
Marcus V
MV
Romano-Silva
Marco A
MA
D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D012604:Scorpion Venoms / Q000032:analysis*; D015222:Sodium Channels / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods
D005456:Fluorescent Dyes; D012604:Scorpion Venoms; D015222:Sodium Channels