PHOSPHATASE ANTAGONIST OKADAIC ACID INHIBITS STEADY-STATE K+ CURRENTS IN GUARD-CELLS OF VICIA-FABA

The effects of protein phosphatase inhibitors on steady-state K+ currents in the plasma membrane of Vicia faba guard cells were studied. Cells were impaled with double barrelled electrodes to monitor membrane voltage and K+ currents under voltage clamp. Okadaic acid (OA) (1 mu M), a specific inhibitor of phosphatase 1 and 2A activity, blocks inward, (I-K+(in)) and outward (I-K+(out)) rectifying K+ channels. Both currents decreased in parallel with a sigmoidal time course with 50% inhibition at about 8 min. With 0.2 mu M OA inhibition became slower and more variable (4-34 min). Inhibition did not recover by washing cells less than or equal to 20 min in OA-free solution. In five out of seven cells OA also induced a rise in the background conductance, which lagged behind the inhibition of K+ current. Both decaying I-K+(out) and rising leak conductance caused a depolarization. OA-induced inhibition of I-K+(in) and I-K+(out) was without a significant effect on the kinetics of voltage-dependent current activation and deactivation. In an alternative approach, guard cells were loaded from the voltage recording pipette with the non-specific phosphatase inhibitor naphthylphosphate. After an impalement of some minutes I-K+(in) and I-K+(out) were small or undetectable. In conclusion inward and outward K+ channels in guard cells have a common voltage-independent mode of control which is sensitive to phosphatase inhibitors. The known specificity of OA points to a mode of action in which a net increase of protein phosphorylation through inhibition of phosphatase 1 and/or 2A activity blocks conductance of both, I-K+(in) and I-K+(out).