Gilliham Matthew, Sullivan Wendy, Tester Mark, Tyerman Stephen D
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK.
Plant J. 2006 Apr;46(1):134-44. doi: 10.1111/j.1365-313X.2006.02676.x.
We present a thorough calibration and verification of a combined non-invasive self-referencing microelectrode-based ion-flux measurement and whole-cell patch clamp system as a novel and powerful tool for the study of ion transport. The system is shown to be capable of revealing the movement of multiple ions across the plasma membrane of a single protoplast at multiple voltages and in complex physiologically relevant solutions. Wheat root protoplasts are patch clamped in the whole-cell configuration and current-voltage relations obtained whilst monitoring net K+ and Ca2+ flux adjacent to the membrane with ion-selective electrodes. At each voltage, net ion flux (nmol m(-2) sec(-1)) is converted to an equivalent current density (mA m(-2)) taking into account geometry and electrode efficiency, and compared with the net current density measured with the patch clamp system. Using this technique, it is demonstrated that the K+-permeable outwardly rectifying conductance (KORC) is responsible for net outward K+ movement across the plasma membrane [1:1 flux-to-current ratio (1.21 +/- 0.14 SEM, n = 15)]. Variation in the K+ flux-to-current ratio among single protoplasts suggests a heterogeneous distribution of KORC channels on the membrane surface. As a demonstration of the power of the technique we show that despite a significant Ca2+ permeability being associated with KORC (analysis of tail current reversal potentials), there is no correlation between Ca2+ flux and KORC activity. A very significant observation is that large Ca2+ fluxes are electrically silent and probably tightly coupled to compensatory charge movements. This analysis demonstrates that it is mandatory to measure flux and currents simultaneously to investigate properly Ca2+ transport mechanisms and selectivity of ion channels in general.
我们展示了一种基于非侵入性自参比微电极的离子通量测量与全细胞膜片钳相结合系统的全面校准和验证，该系统是研究离子转运的一种新颖且强大的工具。结果表明，该系统能够揭示多种离子在多个电压下以及在复杂的生理相关溶液中跨单个原生质体质膜的移动情况。将小麦根原生质体以全细胞模式进行膜片钳操作，并在通过离子选择性电极监测膜附近净K⁺和Ca²⁺通量的同时获得电流 - 电压关系。在每个电压下，考虑到几何形状和电极效率，将净离子通量（nmol m⁻² s⁻¹）转换为等效电流密度（mA m⁻²），并与膜片钳系统测量的净电流密度进行比较。使用该技术表明，K⁺通透外向整流电导（KORC）负责K⁺跨质膜的净外向移动[通量与电流比为1:1（1.21±0.14 SEM，n = 15）]。单个原生质体之间K⁺通量与电流比的变化表明KORC通道在膜表面呈异质分布。作为该技术强大功能的一个例证，我们表明尽管KORC与显著的Ca²⁺通透性相关（对尾电流反转电位的分析），但Ca²⁺通量与KORC活性之间没有相关性。一个非常重要的观察结果是，大的Ca²⁺通量在电学上是沉默的，并且可能与补偿性电荷移动紧密耦合。该分析表明，为了全面研究Ca²⁺转运机制和一般离子通道的选择性，必须同时测量通量和电流。
