Calcium channels in the vacuolar membrane of plants: multiple pathways for intracellular calcium mobilization

An increasing number of studies imply that Ca 2+ mobilization from intracellular stores plays an important role in stimulus evoked elevation of cytosolic free calcium during signal transduction in plants. It is believed that Ca 2+ is released mainly from the vacuole, which contains a high Ca 2+ concentration in a large volume, and can be regarded as the principal Ca 2+ pool in mature higher plant cells. The large size of the organelle confers unique experimental advantages to the study of endomembrane ion channels. The patch-clamp technique can be directly applied to isolated vacuoles to characterize Ca 2+ release pathways at the single channel level and confirm their membrane location. Using radiometric, ligand-binding and electrophysiological techniques we characterized two different pathways by which Ca 2+ can be mobilized from the vacuole of Beta vulgaris tap roots. Inositol 1,4,5 trisphosphate (Ins P 3 )-elicited Ca 2+ release from tonoplast enriched vesicles is dose-dependent, highly specific for Ins P 3 , and is competitively inhibited by low M r heparin ( K i = 34 nM). This striking resemblance to the animal counterpart which is probably located in the ER is further reflected by the binding properties of the solubilized Ins P 3 receptor from beet, which bears similarities to the Ins P 3 receptor of cerebellum. Thus, Ins P 3 and heparin bind to a single site with sub-micromolar K d s, whereas other inositol phosphates have affinities in the supra-micromolar range. The second Ca 2+ channel in the beet tonoplast is voltage-sensitive and channel openings are largely promoted by positive shifts in the vacuolar membrane potential over the physiological range. Channel activity is neither affected by Ins P 3 addition nor by alteration of cytosolic free calcium, and from a large range of Ca 2+ antagonists tested, only Zn 2+ and the lanthanide Gd 3+ proved to be effective inhibitors. With Ca 2+ as a charge carrier the maximum unitary slope conductance is about 12 pS and saturation occurs at < 5 mM vacuolar Ca 2+ . The channel has an approximately 20-fold higher selectivity for Ca 2+ over K + which is achieved by a Ca 2+ binding site in the channel pore. The unique properties of this novel Ca 2+ release pathway suggests that it is specific for plants. The presence of both Ins P 3 -gated and voltage-gated Ca 2+ channels at the vacuolar membrane implies flexibility in the mechanism of intracellular Ca 2+ mobilization in plant cells.