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.
RAVE and Rabconnectin-3 Complexes as Signal Dependent Regulators of Organelle Acidification