Zhang Wei, Nilson Sarah E, Assmann Sarah M
Biology Department, Penn State University, University Park, PA 16802-5301, USA.
CSH Protoc. 2008 Jun 1;2008:pdb.prot5014. doi: 10.1101/pdb.prot5014.
INTRODUCTIONThe flux of ions across membranes via ion channels is vital to cellular responses to internal and external stimuli, and therefore to cellular survival in changing circumstances. Patch clamping is a powerful technique for ion channel investigation, because it enables measurement of both net ion fluxes across the entire surface area of a cell and ion currents flowing through a single open channel. However, unlike animal cells, plant cells are surrounded by cell walls that prevent the physical contact between the patch pipette and the plasma membrane necessary for the patch clamp technique. To demonstrate how patch clamping can be applied to plant physiology research, we describe a protocol used to record potassium ion (K(+)) channel currents in Arabidopsis guard cell protoplasts (a widely studied model cell type in plant biology). The protocol requires a two-step cellulase and pectinase digestion to isolate high quality Arabidopsis guard cell protoplasts (i.e., plant cells lacking their cell walls), preparation of suitable glass capillary microelectrodes, and formation of the whole-cell configuration with a gigaohm (GΩ) seal. We also describe the history of the protocol and list other types of plant cells from which successful patch clamp recordings have been obtained.
引言
离子通过离子通道跨膜流动对于细胞对内部和外部刺激的反应至关重要，因此对于细胞在不断变化的环境中的存活也至关重要。膜片钳技术是研究离子通道的一种强大技术，因为它能够测量跨细胞整个表面积的净离子通量以及流经单个开放通道的离子电流。然而，与动物细胞不同，植物细胞被细胞壁包围，这阻碍了膜片钳技术所需的膜片吸管与质膜之间的物理接触。为了证明膜片钳技术如何应用于植物生理学研究，我们描述了一种用于记录拟南芥保卫细胞原生质体（植物生物学中广泛研究的一种模型细胞类型）中钾离子（K⁺）通道电流的实验方案。该方案需要两步纤维素酶和果胶酶消化以分离高质量的拟南芥保卫细胞原生质体（即没有细胞壁的植物细胞），制备合适的玻璃毛细管微电极，并形成具有千兆欧（GΩ）封接的全细胞配置。我们还描述了该方案的历史，并列出了已成功进行膜片钳记录的其他类型的植物细胞。
