K+ CHANNELS OF STOMATAL GUARD-CELLS - CHARACTERISTICS OF THE INWARD RECTIFIER AND ITS CONTROL BY PH

Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (K(o)+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on K(o)+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ > Rb+ > Cs+ >> Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (K(d) << 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of K(o)+, and the current activated only with supramillimolar K+ outside (E(K)+ > -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pH(o) from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pH(o), but was blocked reversibly by acid-going intracellular pH. The action of pH(o) on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less-than-or-equal-to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pH(o) raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pK(a) approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside. The high sensitivity of the K+ inward rectifier to [H+]o presents a mechanistic basis for understanding K+ channel and H+-ATPase integration over a wide range of environmental conditions, and the H+ sensitivities of both K+ inward and outward rectifiers intimate roles for pH in controlling K+ flux during stomatal movements.