Tonoplast anion channel activity modulation by pH in Chara corallina

The patch-clamp technique was used to investigate regulation of anion chann el activity in the tonoplast of Chara corallina in response to changing pro ton and calcium concentrations on both sides of the membrane. These channel s are known to be Ca2+-dependent, with conductances in the range of 37 to 4 8 pS at pH 7.4. By using low pH at the vacuolar side (either pH(vac) 5.3 or 6.0) and a cytosolic pH (pH(cyt)) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulat ed. In addition, Ca2+- sensitivity of the channels was markedly influenced by pH changes. At pH(cyt) values of 7.2 and 7.4 the half-maximal concentrat ion (EC50) for calcium activation was 100-200 muM, whereas an Ec(50) of abo ut 5 muM was found at a pH,yt of 6.0. This suggests an improved binding of Ca2+ ions to the channel protein at more acidic cytoplasm. At low pH(cyt), anion channel activity and mean open times were voltage-dependent. At pipet te potentials (V-P) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean open time of the channel increased. In contrast, at pH(cyt) 7.2, anion channel activi ty and the opening behavior seemed to be independent of the applied V-P. Th e kinetics of the channel could be further controlled by the Ca2+ concentra tion at the cytosolic membrane side: the mean open time significantly incre ased in the presence of a high cytosolic Ca2+ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range, below the resting pH(cyt). A putative physiological r ole of the pH-dependent modulation of these anion channels is discussed.