Transmembrane potential measurements on plant cells using the voltage-sensitive dye ANNINE-6

Abstract

The charging of the plasma membrane is a necessary condition for the generation of an electric-field-induced permeability increase of the plasmalemma, which is usually explained by the creation and the growth of aqueous pores. For cells suspended in physiological buffers, the time domain of membrane charging is in the submicrosecond range. Systematic measurements using Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) protoplasts stained with the fast voltage-sensitive fluorescence dye ANNINE-6 have been performed using a pulsed laser fluorescence microscopy setup with a time resolution of 5 ns. A clear saturation of the membrane voltage could be measured, caused by a strong membrane permeability increase, commonly explained by enhanced pore formation, which prevents further membrane charging by external electric field exposure. The field strength dependence of the protoplast's transmembrane potential V (M) shows strong asymmetric saturation characteristics due to the high resting potential of the plants plasmalemma. At the pole of the hyperpolarized hemisphere of the cell, saturation starts at an external field strength of 0.3 kV/cm, resulting in a measured transmembrane voltage shift of ∆V(M) = -150 mV, while on the cathodic (depolarized) cell pole, the threshold for enhanced pore formation is reached at a field strength of approximately 1.0 kV/cm and ∆V(M) = 450 mV, respectively. From this asymmetry of the measured maximum membrane voltage shifts, the resting potential of BY-2 protoplasts at the given experimental conditions can be determined to V(R) = -150 mV. Consequently, a strong membrane permeability increase occurs when the membrane voltage diverges |V(M)| = 300 mV from the resting potential of the protoplast. The largest membrane voltage change at a given external electric field occurs at the cell poles. The azimuthal dependence of the transmembrane potential, measured in angular intervals of 10° along the circumference of the cell, shows a flattening and a slight decrease at higher fields at the pole region due to enhanced pore formation. Additionally, at the hyperpolarized cell pole, a polarization reversal could be observed at an external field range around 1.0 kV/cm. This behavior might be attributed to a fast charge transfer through the membrane at the hyperpolarized pole, e.g., by voltage-gated channels.

Basic Features of a Cell Electroporation Model: Illustrative Behavior for Two Very Different Pulses

“…Flattening is observed experimentally (Kinosita et al 1988; Frey et al 2006; Flickinger et al 2010), and is also exhibited by cell membrane models and has been found in cell model responses for both spherical (DeBruin and Krassowska 1999a; Smith and Weaver 2008; Talele et al 2010; Sadik et al 2013), and cylindrical membranes (Stewart et al 2004; Gowrishankar et al 2006; Smith et al 2013). Figure 2b, h shows the model’s angular response for the two pulses.…”

Aspects of plant plasmalemma charging induced by external electric field pulses

“…1 Enhanced pore formation and the associated limitation of a further membrane charging has been detected at the hyperpolarized pole first and also at the depolarized cell pole at higher electric field strengths. A further increase of the field strength resulted in a larger angular range of permeabilized …”

“…If the membrane voltage of a protoplast reaches a value of about ± 300 mV increased membrane permeability has been observed, 1 allowing ions and macromolecules to pass the membrane. This transmembrane current prevents further membrane charging and causes a saturation of the membrane voltage.…”

“…4 The detailed experimental setup and procedure is described in elsewhere. 1 Recharging of the Plasmalemma BY-2 protoplasts stained by ANNINE-6 have been exposed to electric field pulse trains of different amplitudes and the fluorescence intensity change has been recorded at Dt = 500 ns after onset of a 1 ms long electric field pulse. Each field pulse application was followed by a control measurement without external electric field.…”

Cellular physics

“…Several theoretical, but competing, models have been proposed to explain this phenomenon, but experimental evidence was lacking, at least for plant cells. In the current issue, Flickinger et al (2010) have used a newly developed Pulsed Laser Fluorescence Microscopy setup in combination with the voltage-sensitive fluorescent dye ANNINE-6 to follow membrane charging with an unprecedented time resolution of 5 ns in tobacco BY-2 protoplasts. They can show maximal voltage changes at the cell poles and can depict the geometrical distribution of charge revealing a distinct azimuthal pattern.…”