Effects of electric fields on the membrane enzymes of plant cells

本课题旨在研究外加电场对植物细胞质膜酶H-ATP酶在体内催化功能的影响。我们采用膜片钳技术监测植物细胞质膜H-ATP酶的质子泵活性。为了能够通过膜片钳技术检测质子泵活性作为H电流，我们必须找到离子通道介导的电流与质膜中H-ATP酶介导的H电流相比小到可以忽略不计的条件。另一方面，完整细胞的膜电位提供了其质膜中H-ATP酶的体内活性的有用指标。因此，本课题需要发展一种简便的测定原生质体膜电位的方法。1.应用膜片钳技术对烟草原生质体细胞质膜上离子通道的特性进行了详细的研究。这是sh ...更多信息 认为质膜上存在两种类型的离子通道，K和C1通道，这些通道只能在跨膜电位的去极化范围内打开，并且K通道对碱金属阳离子的渗透性增加的顺序为Cs<Na-Li<K。当跨膜电位被钳位在超极化范围内，除了fusicoccin，这是已知的激活质膜H-ATP酶在植物细胞，产生一个短暂的外向电流在全细胞配置。这种瞬态电流的表征工作正在进行中。2.本文根据西姆斯等提出的机理，导出了加入细胞悬液中的3，3 '-二丙基硫代二氮杂双环菁（dis-C3-（5））的荧光响应与细胞膜电位之间关系的理论表达式，并提出了用这种方法测定细胞膜电位时应满足的条件。从紫草培养细胞中分离的原生质体的实验结果与理论预测一致。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