Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+](i)) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+](i) in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating, To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P-o) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed stales of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state, Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage, We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rights reserved.