%0 Journal Article %1 Zimmermann2006 %A Zimmermann, D. %A Terpitz, U. %A Zhou, A. %A Reuss, R. %A Mueller, K. %A Sukhorukov, V. L. %A Gessner, P. %A Nagel, G. %A Zimmermann, U. %A Bamberg, E. %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2006 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS %K terpitz vladimir %N 2 %P 673-681 %T Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system %U http://dx.doi.org/10.1016/j.bbrc.2006.07.112 %V 348 %X Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.

@article{Zimmermann2006, abstract = {Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.}, added-at = {2011-03-02T09:35:06.000+0100}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, affiliation = {Bamberg, E (Reprint Author), Max Planck Inst Biophys, Dept Biophys Chem, Max Von Laue Str 3, D-60438 Frankfurt, Germany. Max Planck Inst Biophys, Dept Biophys Chem, D-60438 Frankfurt, Germany. Univ Frankfurt, Inst Biophys Chem Biochem Chem \& Pharm, D-60438 Frankfurt, Germany. Univ Wurzburg, Lehrstuhl Biotechnol, D-97074 Wurzburg, Germany. Univ Wurzburg, Lehrstuhl Mol Pflanzenphysiol \& Biophys, D-97082 Wurzburg, Germany.}, author = {Zimmermann, D. and Terpitz, U. and Zhou, A. and Reuss, R. and Mueller, K. and Sukhorukov, V. L. and Gessner, P. and Nagel, G. and Zimmermann, U. and Bamberg, E.}, author-email = {ernst.bamberg@mpibp-frankfurt.mpg.de}, biburl = {https://www.bibsonomy.org/bibtex/21ecc15839841b5307ec859f42f1ea2b2/reichert}, doc-delivery-number = {080UW}, groups = {public}, interhash = {a51c925831ac69ad3d7ea222e0659b15}, intrahash = {1ecc15839841b5307ec859f42f1ea2b2}, issn = {0006-291X}, journal = {BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, journal-iso = {Biochem. Biophys. Res. Commun.}, keywords = {terpitz vladimir}, keywords-plus = {SWELLING-ACTIVATED PATHWAYS; HAMSTER OVARY CELLS; ERYTHROCYTE-GHOSTS; MAMMALIAN-CELLS; FUSION; MEMBRANE; OPTIMIZATION; PROTOPLASTS; LYMPHOCYTES; STRATEGIES}, language = {English}, month = {SEP 22}, number = 2, number-of-cited-references = {27}, pages = {673-681}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, subject-category = {Biochemistry \& Molecular Biology; Biophysics}, times-cited = {5}, timestamp = {2011-03-09T14:16:15.000+0100}, title = {Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system}, type = {Article}, unique-id = {ISI:000240275000048}, url = {http://dx.doi.org/10.1016/j.bbrc.2006.07.112}, username = {reichert}, volume = 348, year = 2006 }