Effects of electric fields on the membrane enzymes of plant cells

本项目的目的是研究外电场对植物细胞质膜H-ATPase体内催化功能的影响。我们利用膜片钳技术来监测植物细胞质膜上H-ATPase的质子泵活性。为了能够用膜片钳技术检测质膜上H电流形式的质子泵活动，必须找到与质膜上H-ATPase介导的H电流相比，离子通道介导的电流小得可以忽略不计的条件。另一方面，完整细胞的膜电位为其质膜中H-ATPase的活性提供了一个有用的指示器。因此，本项目需要开发一种简便的方法来测定原生质体的膜电位。1.利用膜片钳技术对烟草原生质体质膜离子通道特性进行了详细的研究。那是Sh…更重要的是，质膜上存在两种离子通道，K通道和C1通道，这些通道只能在跨膜电位的去极化范围内开放，K通道对碱金属离子的通透性增加的顺序是Cs&lt；Na-Li；K；当跨膜电位被钳制在超极化范围内时，加入能够激活植物细胞质膜H-ATPase的褐霉素，产生了全细胞结构的瞬时外向电流。对这种暂态电流的表征目前正在进行中。2.根据Sims等人提出的机理，推导了加入到细胞悬浮液中的菁染料3，3‘-二丙基硫代二碳菁染料(dis-C3-(5))的荧光响应与细胞膜电位之间关系的理论表达式。提出了用该方法评价膜电位时应满足的条件。理论预测与利用紫草培养细胞原生质体获得的实验结果吻合较好。较少

The aims of the present project is to study the effects of external electric fields on the in vivo catalytic functions of the plasma-membrane enzyme, H-ATPase, of plant cells. We employed the patch clamp technique to monitor the proton-pumping activity of H-ATPase in the plasma membranes of plant cells. In order to be able to detect the proton-pumping activity as H current by the patch clamp technique, we must find the conditions under which the currents mediated by the ion channels are negligibly small compared with the H current mediated by H-ATPase in the plasma membrane. On the other hand, the membrane potentials of intact cells provide a useful indicator of in vivo activity of H-ATPase in their plasma membranes. Thus the present project needs to develop a convenient method for determinig the membrane potential of protoplasts. 1. The properties of ion channels in the plasma membrane of tobacco cultured protoplasts were studied in detail by using the patch clamp technique. It was sh … More own that two types of ion channels, K and C1 channels, are present in the plasma membrane, that these channels can open only in the depolarization range of the transmembrane potential, and that the order of increasing permeability of the K channel to alkali metal cations is Cs<Na-Li<K. When the transmembrane potential was clamped in the hyperpolarization range, addition of fusicoccin, which is known to activate the plasma membrane H-ATPase in plant cells, produced a transient outward current in whole-cell configuration. The characterization of this transient current is now in progress. 2. The theoretical expressions were derived for the relationship between the fluorescence response of the cyanine dye, 3,3'-dipropylthiadicarbocyanine (dis-C3-(5)), added to a cell suspension and the membrane potential of the cell on the basis of the mechanism suggested by Sims et al. The condions which should be satisfied in evaluating the membrane potential by this method are presented. The theoretical predictions agreed well with the experimental results obtained with protplasts isolated from Lithospermum erythrorhizon cultured cells. Less