Mechanisms of fusicoccin action: kinetic modification and inactivation of K(+) channels in guard cells

Abstract

Fusicoccin commonly is thought to promote secondary solute transport via an increase in electrical driving force which follows the enhancement of primary, "electrogenic" H(+) extrusion by the plant plasma membrane H(+)-ATPase. However, previous electrical studies ofVicia faba L. guard cells in FC (Blatt, 1988, Planta174, 187) demonstrated, in addition to a limited rise in pump current, appreciable declines in membrane conductance near and positive to the free-running membrane potential (V m). Much of the current at these potentials could have been carried by outward-rectifying K(+) channels which were progressively inactivated in FC. We have examined this possibility in electrical studies, using whole-cell currents measured under voltage clamp to quantitate steadystate and kinetic characteristics of the K(+) channels both before and during exposure to FC; channels block in tetraethylammonium chloride was exploited to assess changes in background 'leak' currents. The cells showed little evidence of primary pump activity, a fact which further simplified analyses. Under these conditions, outward-directed K(+) channel current contributed to charge balance maintainingV m, and adding 10 μM FC on average depolarized (positive-going)V m. Steady-state current-voltage relations revealed changes both in K(+) channel and in leak currents underlying the voltage response. Changes in the leak were variable, but on average the leak equilibrium potential was shifted (+)19 mV and leak conductance declined by 21% over 30-40 min in FC. Potassium currents were inactivated irreversibly and with halftimes (current maxima) of 6.2-10.7 min. Inactivation was voltage-dependent, so that the activation ("gating") potential for the current was shifted, positive-going, with time in FC. Channel gating kinetics, inferred from the macroscopic currents, were also affected; current rise at positive potentials accelerated 4.5-fold and more, but in a manner apparently independent of voltage and extracellular potassium concentration. Current decay at negative potentials was quickened, also. These results identify the outward-rectifying K(+) channels as one site of action for FC at a higher plant cell membrane; they complete the link introduced in the preceding paper between K(+) channel current, K(+)((86)Rb(+)) flux and irreversible cation uptake in the toxin. The data also offer some insights toward a kinetic description of channel gating. Finally, they provide a vehicle for interpreting FC-induced changes in K(+) and net H(+) flux, and in membrane potential without the necessity for postulating gross changes in H(+) pumping.