The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca2+-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pHvac 5.3 or 6.0) and a cytosolic pH (pHcyt) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In addition, Ca2+-sensitivity of the channels was markedly influenced by pH changes. At pHcyt values of 7.2 and 7.4 the half-maximal concentration (EC 50) for calcium activation was 100–200 μm, whereas an EC 50 of about 5 μm was found at a pHcyt of 6.0. This suggests an improved binding of Ca2+ ions to the channel protein at more acidic cytoplasm. At low pHcyt, anion channel activity and mean open times were voltage-dependent. At pipette 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 pHcyt 7.2, anion channel activity and the opening behavior seemed to be independent of the applied V p. The kinetics of the channel could be further controlled by the Ca2+ concentration at the cytosolic membrane side: the mean open time significantly increased 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 pHcyt. A putative physiological role of the pH-dependent modulation of these anion channels is discussed.
The Journal of Membrane Biology – Springer Journals
Published: Nov 15, 2001
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