title: A critical evaluation of whole cell patch clamp studies on electroporation using the voltage sensitive dye ANNINE-6.
abstract: The patch clamp technique in the whole cell configuration is potentially a powerful tool to investigate electroporation (electric-field-induced membrane permeabilization). Membrane polarization beyond certain threshold voltages leads to a steep conductance increase either indicating field-induced pore formation or being due to patch clamp artifacts (seal resistance breakdown). Protoplasts derived from tobacco culture cell lines (Bright Yellow-2, BY-2; Virginia bright Italian-0, VBI-0) were stained with the voltage-sensitive dye ANNINE-6. After establishing the whole cell patch clamp configuration 50-ms command voltage (Ucomm) steps ranging from -500 mV to +500 mV were applied while simultaneously exposing protoplasts to light at 475 nm wavelength. Pulse-induced currents and fluorescence intensity (known to be linearly related to the trans-membrane voltage, Um) were recorded. Plotting fluorescence intensity against Ucomm revealed saturation of the curve at values<-300 mV and >+300 mV and close correlation with theoretical Um values calculated on the basis of membrane pore formation. For BY-2 and VBI-0 protoplasts ANNINE-6 voltage sensitivity was calculated to be -0.0014 mV(-1) and -0.0012 mV(-1), respectively. Voltage ramp experiments revealed cation-selectivity of field-induced pores. Anions are conducted poorly independent of their size. In conclusion, the patch clamp technique is validated as a useful tool in electroporation research.
authors.affiliation: Institute of Pulsed Power & Microwave Technology, Campus North, Karlsruhe Institute of Technology (KIT), Post Office Box 3640, D-76021 Karlsruhe, Germany. lars.wegner@ihm.fzk.de
authors.forename: Lars H
authors.initials: LH
authors.lastname: Wegner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Frey
authors.forename: Sina
authors.initials: S
authors.lastname: Schönwälder
mesh_terms: D002463:Cell Membrane Permeability / Q000502:physiology*; D002911:Chrysenes / Q000737:chemistry*; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques / Q000379:methods*; D011523:Protoplasts / Q000166:cytology; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D014026:Nicotiana / Q000166:cytology; D056969:Voltage-Sensitive Dye Imaging
chemical_list: C530581:ANNINE-6plus; D002911:Chrysenes; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds