Characterization of the Ca2+-Dependent Cl− Efflux in Perfused Chara Cells

Abstract

The mechanism of the Ca2+-dependent Cl− efflux was studied in tonoplast-free cells, in which the intracellular chemical composition can be freely controlled. Tonoplast-free cells were prepared by perfusing the cell interior of internodal cells of Chara corallina with a medium that contained EGTA. The Ca2+-induced Cl− efflux was measured together with the membrane potential during continuous intracellular perfusion. The dependencies of Cl− efflux and the membrane potential on the intracellular Ca2+ or Cl− concentrations were analyzed. When perfusion was started with medium that contained Ca2+ ions, Cl− efflux and membrane depolarization were induced. The amount of Cl− efflux varied considerably among individual cells. The rate of efflux decreased exponentially but a residual efflux remained detectable. The Cl− efflux was induced at concentrations of Ca2+ ions above 1 μM and reached a maximum at 1 mM. By contrast, the membrane depolarization reached a maximum at about 10 μM Ca2+. The rate of Cl− efflux increased linearly with logarithmic increases in the intracellular Cl− concentrations. These findings suggest that more than two kinds of Ca2+-dependent Cl− channel might be present in the plasma membrane. Addition of ATP or its removal from the perfusion medium did not affect the Ca2+-dependent Cl− efflux. Calmodulin antagonists slightly inhibited the Ca2+-dependent Cl− efflux.