Sukhorukov V L, Endter J M, Zimmermann D, Shirakashi R, Fehrmann S, Kiesel M, Reuss R, Becker D, Hedrich R, Bamberg E, Roitsch Th, Zimmermann U
Lehrstuhl für Biotechnologie, Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Würzburg, Germany.
Biophys J. 2007 Nov 1;93(9):3324-37. doi: 10.1529/biophysj.107.110783. Epub 2007 Aug 3.
Cytosolic Ca(2+) changes induced by electric field pulses of 50-micros duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca(2+)-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca(2+) concentration reaching a peak value within <100-200 ms. Peaking of Ca(2+) was followed by a biphasic decay due to removal of Ca(2+) (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of approximately 0.5 and 3-5 s, respectively. Experiments with various external Ca(2+) concentrations and conductivities showed that the fast decay arises from Ca(2+) fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca(2+) fluxes through the tonoplast. The amplitude of the Ca(2+) 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 approximately 450 V/cm, whereas breakdown of the tonoplast required approximately 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 Ca(2+) and presumably other vacuolar ingredients.
通过测量水母发光蛋白转化的BY - 2烟草细胞中的化学发光，监测了持续时间为50微秒、强度为200 - 800 V/cm的电场脉冲诱导的胞质Ca(2+)变化。在Ca(2+)替代培养基中，电脉冲导致胞质Ca(2+)浓度非常快速地初始增加，在<100 - 200毫秒内达到峰值。Ca(2+)达到峰值后，由于Ca(2+)的去除（例如，通过在细胞质中结合和/或隔离），会出现双相衰减。衰减有快速和缓慢成分，其时间常数分别约为0.5秒和3 - 5秒。对不同外部Ca(2+)浓度和电导率的实验表明，快速衰减源于Ca(2+)通过质膜的通量，而缓慢衰减必定归因于Ca(2+)通过液泡膜的通量。Ca(2+)瞬变的幅度随场强增加而增大，而衰减动力学的时间常数保持不变。在约450 V/cm的临界场强下实现质膜破裂，而液泡膜破裂需要约580 V/cm。上述发现可以通过响应指数衰减的场脉冲在两个膜上产生的瞬态电位分布来解释。计算液泡膜和质膜电位所需的介电数据来自对分离的有液泡和无液泡的BY - 2原生质体的电旋转实验。有液泡原生质体的电旋转响应可以用三壳模型来描述（即假设液泡膜和质膜的电容串联排列）。除其他外，理论分析和实验数据表明，通过电转染或电融合对植物细胞进行基因操作必须在低电导率培养基中进行，以尽量减少液泡Ca(2+)以及可能其他液泡成分的释放。
