Characterization of a G-protein-regulated outward K⁺ current in mesophyll cells of Vicia faba L.

Whole-cell voltage-dependent currents in isolated mesophyll protoplasts of Vicia faba were investigated by patch-clamp techniques. With 104 mM K⁺ in the cytosol and 13 mM K⁺ in the external solution, depolarization of the plasma membrane from -47 mV to potentials between -15 and +85 mV activated a voltage- and time-dependent outward current (I_out). The average magnitude of I_out at +85 mV was 28.5 ± 3.3 pA·pF^-1. No inward voltage-dependent current was observed upon hyperpolarization of the plasma membrane from -55 mV to potentials as negative as -175 mV. Time-activated outward current was blocked by Ba²⁺ (1 mM BaCl₂) and was not observed when K⁺ was eliminated from the external and internal solutions, indicating that this outward current was carried primarily by K⁺ ions. The voltage dependency of outward K⁺ current revealed a possible mechanism for K⁺ efflux from mesophyll cells. A GDP analogue guanosine 5′-[β-thio]diphosphate (500 μM) significantly enhanced out-ward K⁺ current. The outward K⁺ current was inhibited by the GTP analogue guanosine 5′-[γ-thio]triphosphate (500 μM) and by an increase in cytoplasmic free Ca²⁺ concentrations. Cholera toxin, which ADP-ribosylates guanine nucleotide-binding regulatory proteins, also inhibited outward K⁺ current. These findings illustrate the presence in mesophyll cells of outward-rectifying K⁺ channels that are regulated by GTP-binding proteins and calcium.