Characteristics of Anion Channels in the Tonoplast of the

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca2þ activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg2þ and low Ca2þ concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg2þ with Kþ in the bath. Sr2þ and Ba2þ were also effective activators of the currents. With a Cl gradient, 10mM in the bath and 100mM in the lumen, currents were significantly reduced and the current–voltage characteristics shifted towards the reversal potential of Cl, indicating Cl selectivity. Currents almost vanished after substituting Cl with gluconate. They were strongly reduced by anion channel inhibitors 4,40-diisothicyanatostilbene-2,20-disulfonic acid (DIDS; 1mM), anthracene-9-carboxylic acid (A9C; 2mM) and ethacrinic acid (0.5mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conduct-ing anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physio-logical calcium concentration. The channels were almost equally permeable to Cl, NO3 and SO 2 4, and much less permeable to malate2. Anion channels did not respond to ATP addition. cAMP (10 mM) had a weak effect on anion channels. Protein kinase A (0.4U) added to the medium caused no significant effect on anion channels