Whole-cell K(+) current across the plasma membrane of guard cells from a grass: Zea mays

Knowledge of ion fluxes in the dumbell-shaped guard cells of grass species has been limited by the difficulty of obtaining isolated epidermes or guard-cell protoplasts for use in radioactive-tracer or electrophysiological studies. We describe here a method for isolating guard-cell protoplasts from Zea mays L. Whole-cell patch clamp has been used to measure K(+)-channel current across the plasma membrane surrounding these protoplasts. Two populations of K(+)-permeable channels have been identified. Hyperpolarization of the membrane to potentials (Vm) more negative than -100 mV results in inward K(+) current through one population of channels. Inward current activation is faster than in the dicotyledon, Vicia faba L. (mean activation half-time 26 ms (Z. mays) versus 123 ms (V. faba) at Vm=-180 mV). Steady-state current density is less than in V. faba (-22 μA · cm(-2) (Z. mays) versus -40 μA · cm(-2) (V. faba) at Vm=- 180 mV in 12 mM external K(+)). Depolarization of the membrane to potentials more positive than -20 mV results in outward K(+) current through a second population of channels; these channels activate and (upon repolarization of the membrane) deactivate more slowly than in V. faba (mean activation half-time 375 ms (Z. mays) versus 187 ms (V. faba) at Vm=+ 80 mV) but result in a similar steady-state current density (23.8 μA · cm(-2) (Z. mays) versus 28.7 μA · cm(-2) (V. faba) at Vm= + 80 mV with 105 mM internal K(+)). Omission of K(+) eliminates the current. The K(+) current is sensitive to both internal and external Ca(2+) concentration: increasing internal Ca(2+) from 2 nM to 0.2 μM or increasing external Ca(2+) from 1 mM to 8.5 mM reduces the magnitude of both inward and outward current.