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

作者：Wegner Lars H^*; Frey Wolfgang; Schoenwaelder Sina

来源：Bioelectrochemistry, 2013, 92: 42-46.

DOI：10.1016/j.bioelechem.2013.03.002

• Electroporation
• Patch clamp
• Field-induced membrane pores
• Voltage-sensitive dye ANNINE-6
• Ion selectivity

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 (U-comm) 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, U-m) were recorded. Plotting fluorescence intensity against Ucomm revealed saturation of the curve at values %26lt; -300 mV and %26gt; +300 mV and close correlation with theoretical U-m 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.

• 出版日期2013-8