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- cha nnels 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 e xtracellular 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, t hese channels activate at hyperpolarising voltages. We found that the open probability (P-o) increased strongly with external Ba2+ concentration, cons istent with a 4-fold cooperative action of Ba2+ in which its binding promot ed channel opening in the steady state. Dwell time analyses indicated the p resence of a single open state and at least three closed stales of the chan nel, and showed that both hyperpolarising voltage and external Ba2+ concent ration prolonged channel residence in the open state, Remarkably, increasin g Ba2+ concentration also enhanced the sensitivity of the open channel to m embrane voltage, We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the v oltage gate, (C) 2001 Federation of European Biochemical Societies. Publish ed by Elsevier Science B,V, All rights reserved.