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

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.