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.
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.
Gilliham
Matthew
M
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK. mg253@cantab.net
Sullivan
Wendy
W
Tester
Mark
M
Tyerman
Stephen D
SD
D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004553:Electric Conductivity; D017136:Ion Transport; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques / Q000379:methods*; D011188:Potassium / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism*
D011188:Potassium; D002118:Calcium