## Abstract

An increasing number of studies imply that Ca<latex>$^{2+}$</latex> 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<latex>$^{2+}$</latex> is released mainly from the vacuole, which contains a high Ca<latex>$^{2+}$</latex> concentration in a large volume, and can be regarded as the principal Ca<latex>$^{2+}$</latex> 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<latex>$^{2+}$</latex> 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<latex>$^{2+}$</latex> can be mobilized from the vacuole of Beta vulgaris tap roots. Inositol 1,4,5 trisphosphate (InsP<latex>$_{3}$</latex>)-elicited Ca<latex>$^{2+}$</latex> release from tonoplast enriched vesicles is dose-dependent, highly specific for InsP<latex>$_{3}$</latex>, and is competitively inhibited by low M<latex>$_{\text{r}}$</latex> heparin (K<latex>$_{\text{i}}$</latex> = 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 InsP<latex>$_{3}$</latex> receptor from beet, which bears similarities to the InsP<latex>$_{3}$</latex> receptor of cerebellum. Thus, InsP<latex>$_{3}$</latex> and heparin bind to a single site with sub-micromolar K<latex>$_{\text{d}}$</latex>s, whereas other inositol phosphates have affinities in the supra-micromolar range. The second Ca<latex>$^{2+}$</latex> 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 InsP<latex>$_{3}$</latex> addition nor by alteration of cytosolic free calcium, and from a large range of Ca<latex>$^{2+}$</latex> antagonists tested, only Zn<latex>$^{2+}$</latex> and the lanthanide Gd<latex>$^{3+}$</latex> proved to be effective inhibitors. With Ca<latex>$^{2+}$</latex> as a charge carrier the maximum unitary slope conductance is about 12 pS and saturation occurs at <latex>$\leq $</latex> 5 mM vacuolar Ca<latex>$^{2+}$</latex>. The channel has an approximately 20-fold higher selectivity for Ca<latex>$^{2+}$</latex> over K<latex>$^{+}$</latex> which is achieved by a Ca<latex>$^{2+}$</latex> binding site in the channel pore. The unique properties of this novel Ca<latex>$^{2+}$</latex> release pathway suggests that it is specific for plants. The presence of both InsP<latex>$_{3}$</latex>-gated and voltage-gated Ca<latex>$^{2+}$</latex> channels at the vacuolar membrane implies flexibility in the mechanism of intracellular Ca<latex>$^{2+}$</latex> mobilization in plant cells.