title: Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins.
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 to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.
authors.affiliation: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Terpitz
authors.forename: Daniel
authors.initials: D
authors.lastname: Raimunda
authors.forename: Markus
authors.initials: M
authors.lastname: Westhoff
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Sukhorukov
authors.forename: Luis
authors.initials: L
authors.lastname: Beaugé
authors.forename: Ernst
authors.initials: E
authors.lastname: Bamberg
authors.forename: Dirk
authors.initials: D
authors.lastname: Zimmermann
mesh_terms: D002462:Cell Membrane; D004594:Electrophysiology; D018408:Patch-Clamp Techniques / Q000379:methods; D011523:Protoplasts / Q000166:cytology*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000502:physiology*