Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Biochimica Et Biophysica Acta
U TerpitzDirk Zimmermann

Abstract

Giant protoplasts of Saccharomyces cerevisiae of 10-35 microm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and "patchability" of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66+/-0.07 microF/cm(2)) and conductance (23-44 microS/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked...Continue Reading

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Citations

Aug 9, 2012·The Journal of Membrane Biology·Ulrich TerpitzVladimir L Sukhorukov
Sep 22, 2012·Assay and Drug Development Technologies·Ulrich TerpitzDirk Zimmermann
Feb 25, 2011·PloS One·Olga N PakhomovaAndrei G Pakhomov
Mar 12, 2008·Biochemical and Biophysical Research Communications·D ZimmermannV L Sukhorukov
Feb 24, 2015·Analytical and Bioanalytical Chemistry·Shi-Yang TangKhashayar Khoshmanesh
Jan 13, 2018·International Journal of Molecular Sciences·Alexander AdamUlrich Terpitz
Nov 12, 2020·International Journal of Molecular Sciences·Francisca Lidiane Linhares de AguiarSónia Gonçalves

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