A critical evaluation of whole cell patch clamp studies on electroporation using the voltage sensitive dye ANNINE-6

• In the whole cell configuration ms pulsed electric fields (PEFs) were imposed.
• Membrane potential (MP) during pulsing was recorded with fluorescent dye (ANNINE-6).
• MP saturation at extremely polarizing PEFs is at variance with patch clamp artifact.
• Anion conductance of field-induced pores is poor, independent of anion size.
• Further usage of the patch clamp method in electroporation research is encouraged.

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.