Giant cells obtained by electrofusion: a novel heterologous expression system

TERPITZ, ULRICH; RAIMUNDA, DANIEL; ZHOU, A; FELDBAUER, K; SUKHORUKOV, VLADIMIR L; BEAUGÉ, LUIS; BAMBERG, ERNST; ZIMMERMANN, DIRK

Congreso; 6th EBSA & British Biophysical Society Congress; 2007

Giant HEK293 cells (30-65 µm diameter) and giant protoplasts of the yeasts S. cerevisiae (15-50 µm diameter) and P. pastoris (10-35 µm diameter) were produced by three-dimensional electrofusion1 in hypoosmolar sorbitol media. This large size allowed to apply both whole-cell and giant excised patch-clamp techniques2. Electrophysiological measurements were made at an external osmolaritiy of 270 mOsm in HEK293 giant cells and 850 mOsm in S. cerevisiae electrofused protoplasts. Under these conditions the specific membrane capacitance was 1.1±0.1 µF/cm2 and 0.7±0.1 µF/cm2, respectively. When fluorescein was included in the patch-pipettes a uniform intracellular distribution of the dye was seen in the whole-cell but not in the cell-attached configurations. This excludes internal compartmentalisation of the giant cells and protoplasts. The existence of a homogenous fusion in HEK293 cells was additionally supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein CH2R-YFP) that showed no plasma-membrane bound YFP mediated fluorescence in the interior of the giant HEK293 cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) in HEK293 cells yielded results similar to those reported in the literature. Therefore, this fusion technique can provide heterologeous expression systems suitable to perform functional studies on membrane proteins.