K+-sensitive gating of the K+ outward rectifier in Vicia guard cells

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1-30 mM K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of -200 mV, clamp steps to voltages between -150 and +50 mV in 0.1 mM K+ activated current through outward-rectifying K channels (I-K,I-out) at the plasma membrane in a voltage-dependent fashion. increasing [K+](o) shifted the voltage-sensitivity of I-K,I-out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+](o) was evident in the kinetics of I-K,I-out activation and deactivation, as well as the steady-state conductance-(g(K-)) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near -130 mV in 0.1 mM K+, -80 mV in 1.0 mM K+, and -20 mV in 10 mM K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+ greater than or equal to Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mM was without effect on the voltage-dependence of g(K) or on I-K,I-out activation kinetics, although 10 mM [Ca2+](o) accelerated current deactivation at voltages negative of -75 mV. At any one voltage, increasing [K+](o) suppressed g(K) completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mM with clamp voltages near -100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of I-K,I-out gating; the results implicate K+-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K+ ions outside affects the distribution between closed states of the channel.