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|Title:||Simultaneous flux and current measurement from single plant protoplasts reveals a strong link between K⁺ fluxes and current, but no link between Ca²⁺ fluxes and current|
|Other Titles:||Simultaneous flux and current measurement from single plant protoplasts reveals a strong link between K(+) fluxes and current, but no link between Ca(2+) fluxes and current|
|Citation:||Plant Journal, 2006; 46(1):134-144|
|Publisher:||Blackwell Publishing Ltd|
|Matthew Gilliham, Wendy Sullivan, Mark Tester, Stephen D. Tyerman|
|Abstract:||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.|
|Keywords:||Non-invasive self-referencing microelectrode ion-flux measurement; ion-selective electrodes; Microelectrode Ion-Flux Estimation; patch clamp electrophysiology; ion channels; selectivity|
|Appears in Collections:||Agriculture, Food and Wine publications|
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