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

Matthew Gilliham*, Wendy Sullivan, Mark Tester, Stephen D. Tyerman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

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 Ca 2+ 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 Ca 2+ transport mechanisms and selectivity of ion channels in general.

Original languageEnglish (US)
Pages (from-to)134-144
Number of pages11
JournalPlant Journal
Volume46
Issue number1
DOIs
StatePublished - Apr 1 2006

Keywords

  • Ion channels
  • Ion-selective electrodes
  • Microelectrode Ion-Flux Estimation
  • Non-invasive self-referencing microelectrode ion-flux measurement
  • Patch clamp electrophysiology
  • Selectivity

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

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