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Mechanisms of Electrically Mediated Cytosolic Ca²⁺ Transients in Aequorin-Transformed Tobacco Cells

V. L. Sukhorukov ^*, J. M. Endter ^*, D. Zimmermann , R. Shirakashi , S. Fehrmann ^*, M. Kiesel ^*, R. Reuss ^*, D. Becker , R. Hedrich , E. Bamberg , Th. Roitsch ^¶ and U. Zimmermann ^*

^* Lehrstuhl für Biotechnologie, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften, and ^¶ Institut für Pharmazeutische Biology, Universität Würzburg, Würzburg, Germany; Abteilung für Biophysikalische Chemie, Max-Planck-Institut für Biophysik, Frankfurt, Germany; and Institute of Industrial Science, The University of Tokyo, Tokyo, Japan

Correspondence: Address reprint requests to Ulrich Zimmermann, University of Wuerzburg, Biotechnology Am Hubland, Biozentrum, Wuerzburg D-97074, Germany. Tel.: 49-931-8884508; E-mail: zimmerma{at}biozentrum.uniwuerzburg.de.

Cytosolic Ca²⁺ changes induced by electric field pulses of 50-µs duration and 200–800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca²⁺-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca²⁺ concentration reaching a peak value within <100–200 ms. Peaking of [Ca²⁺]_cyt was followed by a biphasic decay due to removal of Ca²⁺ (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of 0.5 and 3–5 s, respectively. Experiments with various external Ca²⁺ concentrations and conductivities showed that the fast decay arises from Ca²⁺ fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca²⁺ fluxes through the tonoplast. The amplitude of the [Ca²⁺]_cyt transients increased with increasing field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical field strength of 450 V/cm, whereas breakdown of the tonoplast required 580 V/cm. The above findings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca²⁺ and presumably other vacuolar ingredients.