BACKGROUND
the contractile vacuole  network of dictyostelium consists of tubes and bladders.
it transiently fuses with the plasma membrane to expel water and ions and thereby serves as an efficient osmoregulatory organelle  <cit> .
the cv-system is also assumed to be involved in ca2+-transport since it contains a pmca-type ca2+-atpase , calmodulin  <cit>  and a vacuolar proton pump that establishes a proton gradient for ca2+-transport  <cit> .
pat <dig> is localized to the cv and the plasma membrane and is upregulated under conditions of ca2+-stress  <cit> .
downregulation of pat <dig> by antisense rna reduced vesicular ca2+-uptake.
we are interested in the characterization of ca2+-stores that are involved in camp-induced ca2+-influx.
previously, it has been shown that acidic ca2+-stores and an ip3-sensitive store participate in this regulation  <cit> .
acidic means that the stores are equipped with a v-type h+-atpase.
acidic vesicular ca2+-stores in dictyostelium comprise the cv-system, endosomes and acidocalcisomes  <cit> .
in the present study we focus on the contribution of the cv-system to intracellular ca2+ regulation.
it has been shown previously that gfp-tagged dajumin labels the entire cv whereas the endosomal compartments are devoid of the label  <cit> .
by contrast, drainin, a peripheral membrane protein involved in discharge of the bladder, is found only on the bladder  <cit> .
we used dajumin-gfp expressing cells to obtain a fraction enriched in cv membranes and used this fraction to measure ca2+-transport.
ca2+-transport activity increased with enhanced purity of the cv.
we also employed a lvsa minus strain which lacks the gene for the protein large volume spherea .
besides its involvement in cytokinesis  <cit>  it is known that the lvsa-protein is localized to the cv.
this association with the cv occurs only during the discharge phase of the vacuole.
in the lvsa mutant calmodulin was lost from the cv-membranes and the cv became disorganized, unable to discharge its contents  <cit> .
we found that vesicular ca2+-transport in the lvsa-mutant was diminished and that camp-induced ca2+-influx was drastically reduced, indicating that functional cv are absolutely required for the camp-dependent ca2+-influx.
RESULTS
distribution of cv in vesicular fractions
we used differentiated cells  <dig> to  <dig> hrs after starvation for preparation of ca2+-transporting vesicles because camp-induced ca2+-influx is present at that time and endosomal content is low .
cells labeled with dajumin-gfp as a marker for the cv-system or with calnexin-gfp cells as a marker for the endoplasmic reticulum  are shown in figure  <dig>  dajumin-gfp allows to visualize the dynamics of the bladder by formation of irregular ventricles and ducts .
the er is prominently labeled in the perinuclear region and close to the plasma membrane .
the cells were lysed by passage through nuclepore filters.
vesicular fractions were separated by differential centrifugation and assayed for ca2+-transporting activity.
the dajumin-gfp label was detected in vesicular fractions with the majority being present in the fast sedimenting fraction p <dig> .
by contrast, most of the er occurred in p <dig>  whereas the lightest fraction p <dig> contained only a quarter of both organelles .
plasma membranes, as shown previously, sedimented in p <dig>  <cit> .
ca2+-transport activity was strikingly concentrated in p <dig>  the presence of endosomes was measured with ritc-dextran.
in two independent experiments  <dig> ± 6% of the label was associated with p <dig>   <dig> ± 6% with p <dig> and none was present in p <dig>  however, the amount of endosomes of  <dig> hour starved cells was low and barely detectable.
this result is expected since the cells develop in the absence of nutrients.
if endosomes accumulate ca2+ their contribution to ca2+-transport was therefore considered to be insignificant under the present experimental conditions.
purification of cv with magnetic antibody-beads
since p <dig> was the richest source of cv we incubated p <dig> isolated from dajumin-gfp labeled cells with anti-gfp-magnetic beads and loaded the mixture onto a column.
during loading and washing the beads were retained on the column by applying a magnetic field.
elution was performed in its absence.
as shown in table  <dig> almost all of the dajumin-label was present in the eluate  as well as most of ca2+-uptake.
the flow through  still contained some ca2+-transport activity the percentage of which, however, exceeded only slightly the remaining dajumin-gfp-label.
the selectivity of the gfp-antibody beads for gfp was tested by assaying p <dig> from unlabeled ax2-cells, the parent strain of dajumin-gfp expressing cells, for binding to the anti-gfp-beads.
in three independent experiments  <dig> ±  <dig> μg  protein was associated with p0e in the controls whereas  <dig> ±  <dig> μg  protein was present in p0e from dajumin-gfp labeled cells.
this result shows that the gfp-antibody specifically targets the gfp-tagged protein and that 28% of unspecific binding does occur.
in order to avoid contamination of p <dig> and subsequent fractions with plasma membranes due to possible association of plasma membranes and cv by cortical actin filaments we preincubated the cells  with latrunculin b. latrunculin b, a unique marine toxin, inhibits actin polymerization and disrupts microfilament organization  <cit> .
in the presence of latrunculin b the cells rounded up and were completely immotile before they were pressed through the filters.
the characteristics of ca2+-transport, its inhibition by drugs as well as ca2+-release in p0e were in the same range as in preparations performed without latrunculin b treatment.
latrunculin b treatment reduced the protein content of p0e by a factor of  <dig>  but there was no statistically significant difference in the specific activity of the ca2+-atpase.
a specific marker for the cv as compared to the plasma membrane is the v-type h+-atpase.
antibodies to the 37/ <dig> kda subunit revealed that the proton pump was markedly enriched in p0e .
a mitochondrial contribution to ca2+-fluxes was prevented by inclusion of mitochondrial inhibitors in all vesicular ca2+-transport measurements.
purification of er with magnetic antibody-beads
since p <dig> was the richest source of er  we incubated p <dig> isolated from calnexin-gfp labeled cells with anti-gfp-magnetic beads and separated er-vesicles from other vesicles by a magnetic field.
as shown in table  <dig> almost all of the calnexin-gfp-label was found in the eluate  as well as most of ca2+-uptake activity.
the flow through  also contained some ca2+-transport activity.
the results of table  <dig> thus show that by using the anti-gfp-antibody beads a 6-7-fold enrichment of either cv or er can be obtained.
furthermore, both fractions enriched either in cv or er vesicles, displayed a similar enhancement of ca2+-transport.
this result demonstrates that vesicles of the cv of dictyostelium are functional in ca2+-homeostasis.
properties of cv and er fractions with respect to ca2+-regulation
since both purifications had given a strong enrichment of the gfp-label as well as in ca2+-transport in the eluate  and since in control experiments 28% unlabeled protein was associated with the eluate, we decided to test for the properties of cv and er with respect to ca2+-transport and ca2+-release.
as shown in figure  <dig> p0e, the eluate of dajumin-gfp-labeled cells containing cv, transported ca2+ efficiently.
note, that the slope is fairly steep and uptake of ca2+ is settled in about  <dig> min.
in three independent experiments we found that starting at an extravesicular ca2+-concentration  of  <dig>  ±  <dig>  μm uptake ceased to  <dig>  ±  <dig>  μm 2+ev in the presence of  <dig> ±  <dig> μg protein.
in agreement with previous results obtained for acidic ca2+-stores the calmodulin antagonist w <dig> as well as arachidonic acid  caused ca2+-release in p0e.
the response to w <dig>  amounted to  <dig>  ±  <dig>  nmol ca2+/mg protein  and to aa   <dig>  ±  <dig>  nmol ca2+/mg protein .
in figure  <dig> ca2+-uptake by p1e, the eluate of calnexin-gfp-labeled cells containing er, is shown.
in contrast to p0e p1e was less responsive to aa.
in three out of four experiments no ca2+-release was measured.
the one that responded to aa had a lower purification factor with respect to gfp fluorescence compared with the other three,  <dig>  versus  <dig>  ±  <dig>  .
ip <dig>  most of the times, was inefficient to elicit ca2+-release.
in general, ca2+-uptake was slower in p1e as compared to p0e starting at  <dig>  ±  <dig>  μm 2+ev and ceased to extravesicular ca2+-concentrations of about  <dig>  ±  <dig>  μm in the presence of  <dig> ±  <dig> μg protein , indicating that the cv is more relevant in maintaining a basal cytosolic ca2+-concentration of  <dig> to  <dig> nm  <cit>  than the er.
in addition, we tested for inhibition of ca2+-uptake.
table  <dig> shows that concanamycin a , a powerful inhibitor of v-type h+-atpases  <cit>  inhibited p0e as expected for a compartment that uses a proton gradient for ca2+-transport and a ca2+-transport atpase pat <dig>  under our experimental conditions bhq, an inhibitor of ca2+-transport atpases, inhibited ca2+-uptake only weakly despite its reported property to inhibit camp-induced ca2+-influx in dictyostelium  <cit> .
bhq was more efficient in inhibiting ca2+-uptake into the er-fraction.
the latter was also sensitive to thapsigargin  a blocker of serca-type ca2+-atpases.
in both fractions, p0e and p1e, ca2+-uptake was inhibited by low concentrations  of sr2+ by about 50%.
is the cv involved in camp-induced ca2+-influx?
given our results that the cv is a ca2+-store that transports ca2+ most efficiently at low ca2+-concentrations we wanted to know whether the store is also involved in receptor-mediated ca2+-flux.
although it was previously shown that nbd-cl and cma inhibited the camp mediated ca2+-influx  <cit>  these results only proved that acidic stores as a whole were involved.
we made use of the lvsa--mutant whose cv-function is abolished with respect to fusion of cv with the plasma membrane and is disturbed by a loss of calmodulin.
previously, we have shown that the calmodulin antagonist w <dig> inhibits ca2+-transport of acidic ca2+-stores  <cit> .
a localized loss of calmodulin confined to the cv would have the same consequences.
therefore, it was interesting to know whether or not ca2+-influx was also disturbed in the mutant.
as shown in table  <dig> there was no receptor-mediated ca2+-influx in the lvsa minus strain at physiological camp-concentrations  in contrast to the parent strain dh <dig>  only higher concentrations of camp yielded ca2+-influx that was reduced as compared to dh <dig> .
besides camp, arachidonic acid  was shown to elicit ca2+-influx.
aa acts by eliciting ca2+-release from ca2+-stores which is thought to induce a capacitative ca2+-influx  <cit> .
in the lvsa minus mutant aa did not evoke ca2+-influx in contrast to the parent strain .
instead ca2+-efflux occurred.
in seven independent experiments 6– <dig> μm aa induced an efflux of ca2+ of 276- ±  <dig> pmol/107cells.
analysis of vesicular ca2+-transport in the mutant revealed that transport activity was present in all three fractions of p <dig>  p <dig> and p <dig> albeit at a somewhat lower extent than in ax <dig> .
therefore, the small amount of camp-induced ca2+-influx observed in the lvsa minus strain cannot be due to the lack of vesicular ca2+-transport in the mutant.
discussion
in this study we have shown that one of the acidic compartments of dictyostelium, the cv-system, is required for camp-induced ca2+-influx in intact cells and that isolated cv-vesicles mediate an essential part of ca2+-transport as well as ca2+-release in response to aa.
previously, it was only known that acidic ca2+-stores were involved.
the use of dajumin-gfp-labeled cv allowed a 7-fold enrichment of cv by a specific method utilizing antibodies directed against the gfp-tag.
ca2+-transport was enhanced 5- to 6-fold.
this result is due to ca2+-transport activity of another ca2+-store that was retained in the flow through .
enriched cv displayed a potent and efficient ca2+-transport that resulted in a low basal extravesicular ca2+ concentration.
moreover, the ca2+ taken up could be released by the calmodulin antagonist w- <dig> and by aa.
both compounds have been shown to be potent regulators of the acidic ca2+-stores  <cit> .
in addition, ca2+-uptake was sensitive to cma, an inhibitor of v-type h+-atpases.
moreover, the v-type proton pump was concentrated in p0e.
a similar enrichment was also obtained for the er using calnexin-gfp-labeled cells.
these ca2+-stores differed from those of the cv by a greater sensitivity to bhq, a lower responsiveness to aa and a higher basal extravesicular ca2+-concentration following ca2+-uptake.
in order to examine whether the ca2+-transport occurring in cv is also involved in camp-mediated ca2+-influx we made use of a mutant, lvsa minus.
in this mutant the cv becomes disorganized during the discharge phase of the vacuole and calmodulin dissociates from the cv-membranes.
the cv can swell but seems unable to discharge.
the lvsa-protein that belongs to the family of beach-proteins transiently binds to the cv.
we found that the mutant was strongly impaired in receptor-mediated ca2+-influx when physiological camp-concentrations of  <dig> – <dig> μm and even higher concentrations of  <dig> μm camp were applied.
a significant ca2+-influx was apparent only at elevated camp concentrations of  <dig> to  <dig> μm.
even so, influx was inhibited by about 75% as compared to the parent strain.
other compounds that induce ca2+-influx in dictyostelium are long chain fatty acids like aa.
they act independently of the camp-receptor and of cell differentiation  <cit> .
similarly to camp, aa did not cause ca2+-influx in the lvsa-minus-strain, instead ca2+-efflux occurred.
these results demonstrate that in order to activate capacitative ca2+-influx an intact cv-system is absolutely necessary.
what is the role of the lvsa-protein in ca2+-influx?
the function of the lvsa-protein in dictyostelium is not yet known.
beach-proteins belong to several classes and only a few proteins have been characterized.
the fan-protein mediates tnf-activation via the map-kinase pathway  <cit> .
neurobeachin conveys anchoring of the regulatory subunit of protein kinase a   <cit> .
in both cases, the beach-protein participates in signal transduction that could lead to altered ca2+-regulation in dictyostelium.
elevated camp-levels in dictyostelium were shown to affect both, basal i and stimulus-induced i-transients  <cit> .
apart from this, it was shown that the loss of the lvsa-protein was accompanied by a loss of calmodulin from the cv-membranes.
since we have shown that the calmodulin antagonist w <dig> inhibits ca2+-transport of acidic ca2+-stores  <cit>  we conclude that ca2+-uptake by the cv is inhibited in the absence of calmodulin.
this could be an explanation of the severe reduction of camp-mediated ca2+-influx measured in the lvsa--mutant strain.
CONCLUSIONS
among the acidic ca2+-stores the contractile vacuole represents an efficient ca2+ transport organelle that sequesters ca2+ to low extravesicular ca2+ concentrations and releases ca2+ in response to arachidonic acid.
furthermore, it is essential for the occurrence of receptor-mediated ca2+ influx to physiological concentrations of camp.

 the contractile vacuole network of dictyostelium consists of tubes and bladders. it transiently fuses with the plasma membrane to expel water and ions and thereby serves as an efficient osmoregulatory organelle. the cv-system is also assumed to be involved in ca2+-transport since it contains a pmca-type ca2+-atpase, calmodulin and a vacuolar proton pump that establishes a proton gradient for ca2+-transport. pat is localized to the cv and the plasma membrane and is upregulated under conditions of ca2+-stress. downregulation of pat by antisense rna reduced vesicular ca2+-uptake. we are interested in the characterization of ca2+-stores that are involved in camp-induced ca2+-influx. previously, it has been shown that acidic ca2+-stores and an ip3-sensitive store participate in this regulation. acidic means that the stores are equipped with a v-type h+-atpase. acidic vesicular ca2+-stores in dictyostelium comprise the cv-system, endosomes and acidocalcisomes. in the present study we focus on the contribution of the cv-system to intracellular ca2+ regulation. it has been shown previously that gfp-tagged dajumin labels the entire cv whereas the endosomal compartments are devoid of the label. by contrast, drainin, a peripheral membrane protein involved in discharge of the bladder, is found only on the bladder. we used dajumin-gfp expressing cells to obtain a fraction enriched in cv membranes and used this fraction to measure ca2+-transport. ca2+-transport activity increased with enhanced purity of the cv. we also employed a lvsa minus strain which lacks the gene for the protein large volume spherea. besides its involvement in cytokinesis it is known that the lvsa-protein is localized to the cv. this association with the cv occurs only during the discharge phase of the vacuole. in the lvsa mutant calmodulin was lost from the cv-membranes and the cv became disorganized, unable to discharge its contents. we found that vesicular ca2+-transport in the lvsa-mutant was diminished and that camp-induced ca2+-influx was drastically reduced, indicating that functional cv are absolutely required for the camp-dependent ca2+-influx.  distribution of cv in vesicular fractions we used differentiated cells to hrs after starvation for preparation of ca2+-transporting vesicles because camp-induced ca2+-influx is present at that time and endosomal content is low. cells labeled with dajumin-gfp as a marker for the cv-system or with calnexin-gfp cells as a marker for the endoplasmic reticulum are shown in figure dajumin-gfp allows to visualize the dynamics of the bladder by formation of irregular ventricles and ducts. the er is prominently labeled in the perinuclear region and close to the plasma membrane. the cells were lysed by passage through nuclepore filters. vesicular fractions were separated by differential centrifugation and assayed for ca2+-transporting activity. the dajumin-gfp label was detected in vesicular fractions with the majority being present in the fast sedimenting fraction p. by contrast, most of the er occurred in p whereas the lightest fraction p contained only a quarter of both organelles. plasma membranes, as shown previously, sedimented in p. ca2+-transport activity was strikingly concentrated in p the presence of endosomes was measured with ritc-dextran. in two independent experiments ± 6% of the label was associated with p ± 6% with p and none was present in p however, the amount of endosomes of hour starved cells was low and barely detectable. this result is expected since the cells develop in the absence of nutrients. if endosomes accumulate ca2+ their contribution to ca2+-transport was therefore considered to be insignificant under the present experimental conditions. purification of cv with magnetic antibody-beads since p was the richest source of cv we incubated p isolated from dajumin-gfp labeled cells with anti-gfp-magnetic beads and loaded the mixture onto a column. during loading and washing the beads were retained on the column by applying a magnetic field. elution was performed in its absence. as shown in table almost all of the dajumin-label was present in the eluate as well as most of ca2+-uptake. the flow through still contained some ca2+-transport activity the percentage of which, however, exceeded only slightly the remaining dajumin-gfp-label. the selectivity of the gfp-antibody beads for gfp was tested by assaying p from unlabeled ax2-cells, the parent strain of dajumin-gfp expressing cells, for binding to the anti-gfp-beads. in three independent experiments ± μg protein was associated with p0e in the controls whereas ± μg protein was present in p0e from dajumin-gfp labeled cells. this result shows that the gfp-antibody specifically targets the gfp-tagged protein and that 28% of unspecific binding does occur. in order to avoid contamination of p and subsequent fractions with plasma membranes due to possible association of plasma membranes and cv by cortical actin filaments we preincubated the cells with latrunculin b. latrunculin b, a unique marine toxin, inhibits actin polymerization and disrupts microfilament organization. in the presence of latrunculin b the cells rounded up and were completely immotile before they were pressed through the filters. the characteristics of ca2+-transport, its inhibition by drugs as well as ca2+-release in p0e were in the same range as in preparations performed without latrunculin b treatment. latrunculin b treatment reduced the protein content of p0e by a factor of but there was no statistically significant difference in the specific activity of the ca2+-atpase. a specific marker for the cv as compared to the plasma membrane is the v-type h+-atpase. antibodies to the 37/ kda subunit revealed that the proton pump was markedly enriched in p0e. a mitochondrial contribution to ca2+-fluxes was prevented by inclusion of mitochondrial inhibitors in all vesicular ca2+-transport measurements. purification of er with magnetic antibody-beads since p was the richest source of er we incubated p isolated from calnexin-gfp labeled cells with anti-gfp-magnetic beads and separated er-vesicles from other vesicles by a magnetic field. as shown in table almost all of the calnexin-gfp-label was found in the eluate as well as most of ca2+-uptake activity. the flow through also contained some ca2+-transport activity. the results of table thus show that by using the anti-gfp-antibody beads a 6-7-fold enrichment of either cv or er can be obtained. furthermore, both fractions enriched either in cv or er vesicles, displayed a similar enhancement of ca2+-transport. this result demonstrates that vesicles of the cv of dictyostelium are functional in ca2+-homeostasis. properties of cv and er fractions with respect to ca2+-regulation since both purifications had given a strong enrichment of the gfp-label as well as in ca2+-transport in the eluate and since in control experiments 28% unlabeled protein was associated with the eluate, we decided to test for the properties of cv and er with respect to ca2+-transport and ca2+-release. as shown in figure p0e, the eluate of dajumin-gfp-labeled cells containing cv, transported ca2+ efficiently. note, that the slope is fairly steep and uptake of ca2+ is settled in about min. in three independent experiments we found that starting at an extravesicular ca2+-concentration of ± μm uptake ceased to ± μm 2+ev in the presence of ± μg protein. in agreement with previous results obtained for acidic ca2+-stores the calmodulin antagonist w as well as arachidonic acid caused ca2+-release in p0e. the response to w amounted to ± nmol ca2+/mg protein and to aa ± nmol ca2+/mg protein. in figure ca2+-uptake by p1e, the eluate of calnexin-gfp-labeled cells containing er, is shown. in contrast to p0e p1e was less responsive to aa. in three out of four experiments no ca2+-release was measured. the one that responded to aa had a lower purification factor with respect to gfp fluorescence compared with the other three, versus ±. ip most of the times, was inefficient to elicit ca2+-release. in general, ca2+-uptake was slower in p1e as compared to p0e starting at ± μm 2+ev and ceased to extravesicular ca2+-concentrations of about ± μm in the presence of ± μg protein, indicating that the cv is more relevant in maintaining a basal cytosolic ca2+-concentration of to nm than the er. in addition, we tested for inhibition of ca2+-uptake. table shows that concanamycin a, a powerful inhibitor of v-type h+-atpases inhibited p0e as expected for a compartment that uses a proton gradient for ca2+-transport and a ca2+-transport atpase pat under our experimental conditions bhq, an inhibitor of ca2+-transport atpases, inhibited ca2+-uptake only weakly despite its reported property to inhibit camp-induced ca2+-influx in dictyostelium. bhq was more efficient in inhibiting ca2+-uptake into the er-fraction. the latter was also sensitive to thapsigargin a blocker of serca-type ca2+-atpases. in both fractions, p0e and p1e, ca2+-uptake was inhibited by low concentrations of sr2+ by about 50%. is the cv involved in camp-induced ca2+-influx? given our results that the cv is a ca2+-store that transports ca2+ most efficiently at low ca2+-concentrations we wanted to know whether the store is also involved in receptor-mediated ca2+-flux. although it was previously shown that nbd-cl and cma inhibited the camp mediated ca2+-influx these results only proved that acidic stores as a whole were involved. we made use of the lvsa--mutant whose cv-function is abolished with respect to fusion of cv with the plasma membrane and is disturbed by a loss of calmodulin. previously, we have shown that the calmodulin antagonist w inhibits ca2+-transport of acidic ca2+-stores. a localized loss of calmodulin confined to the cv would have the same consequences. therefore, it was interesting to know whether or not ca2+-influx was also disturbed in the mutant. as shown in table there was no receptor-mediated ca2+-influx in the lvsa minus strain at physiological camp-concentrations in contrast to the parent strain dh only higher concentrations of camp yielded ca2+-influx that was reduced as compared to dh. besides camp, arachidonic acid was shown to elicit ca2+-influx. aa acts by eliciting ca2+-release from ca2+-stores which is thought to induce a capacitative ca2+-influx. in the lvsa minus mutant aa did not evoke ca2+-influx in contrast to the parent strain. instead ca2+-efflux occurred. in seven independent experiments 6– μm aa induced an efflux of ca2+ of 276- ± pmol/107cells. analysis of vesicular ca2+-transport in the mutant revealed that transport activity was present in all three fractions of p p and p albeit at a somewhat lower extent than in ax. therefore, the small amount of camp-induced ca2+-influx observed in the lvsa minus strain cannot be due to the lack of vesicular ca2+-transport in the mutant. discussion in this study we have shown that one of the acidic compartments of dictyostelium, the cv-system, is required for camp-induced ca2+-influx in intact cells and that isolated cv-vesicles mediate an essential part of ca2+-transport as well as ca2+-release in response to aa. previously, it was only known that acidic ca2+-stores were involved. the use of dajumin-gfp-labeled cv allowed a 7-fold enrichment of cv by a specific method utilizing antibodies directed against the gfp-tag. ca2+-transport was enhanced 5- to 6-fold. this result is due to ca2+-transport activity of another ca2+-store that was retained in the flow through. enriched cv displayed a potent and efficient ca2+-transport that resulted in a low basal extravesicular ca2+ concentration. moreover, the ca2+ taken up could be released by the calmodulin antagonist w- and by aa. both compounds have been shown to be potent regulators of the acidic ca2+-stores. in addition, ca2+-uptake was sensitive to cma, an inhibitor of v-type h+-atpases. moreover, the v-type proton pump was concentrated in p0e. a similar enrichment was also obtained for the er using calnexin-gfp-labeled cells. these ca2+-stores differed from those of the cv by a greater sensitivity to bhq, a lower responsiveness to aa and a higher basal extravesicular ca2+-concentration following ca2+-uptake. in order to examine whether the ca2+-transport occurring in cv is also involved in camp-mediated ca2+-influx we made use of a mutant, lvsa minus. in this mutant the cv becomes disorganized during the discharge phase of the vacuole and calmodulin dissociates from the cv-membranes. the cv can swell but seems unable to discharge. the lvsa-protein that belongs to the family of beach-proteins transiently binds to the cv. we found that the mutant was strongly impaired in receptor-mediated ca2+-influx when physiological camp-concentrations of – μm and even higher concentrations of μm camp were applied. a significant ca2+-influx was apparent only at elevated camp concentrations of to μm. even so, influx was inhibited by about 75% as compared to the parent strain. other compounds that induce ca2+-influx in dictyostelium are long chain fatty acids like aa. they act independently of the camp-receptor and of cell differentiation. similarly to camp, aa did not cause ca2+-influx in the lvsa-minus-strain, instead ca2+-efflux occurred. these results demonstrate that in order to activate capacitative ca2+-influx an intact cv-system is absolutely necessary. what is the role of the lvsa-protein in ca2+-influx? the function of the lvsa-protein in dictyostelium is not yet known. beach-proteins belong to several classes and only a few proteins have been characterized. the fan-protein mediates tnf-activation via the map-kinase pathway. neurobeachin conveys anchoring of the regulatory subunit of protein kinase a. in both cases, the beach-protein participates in signal transduction that could lead to altered ca2+-regulation in dictyostelium. elevated camp-levels in dictyostelium were shown to affect both, basal i and stimulus-induced i-transients. apart from this, it was shown that the loss of the lvsa-protein was accompanied by a loss of calmodulin from the cv-membranes. since we have shown that the calmodulin antagonist w inhibits ca2+-transport of acidic ca2+-stores we conclude that ca2+-uptake by the cv is inhibited in the absence of calmodulin. this could be an explanation of the severe reduction of camp-mediated ca2+-influx measured in the lvsa--mutant strain.  among the acidic ca2+-stores the contractile vacuole represents an efficient ca2+ transport organelle that sequesters ca2+ to low extravesicular ca2+ concentrations and releases ca2+ in response to arachidonic acid. furthermore, it is essential for the occurrence of receptor-mediated ca2+ influx to physiological concentrations of camp. 
dajumin-gfp labeled contractile vacuoles were purified 7-fold by anti-gfp-antibodies in a magnetic field. camp-induced ca2+-influx in dictyostelium is controlled by at least two non-mitochondrial ca2+-stores: acidic stores and the endoplasmic reticulum. the acidic stores may comprise the contractile vacuole network, the endosomal compartment and acidocalcisomes. release of ca2+ was elicited by arachidonic acid or the calmodulin antagonist w the latter due to inhibition of the pump. we found a severe reduction of camp-induced ca2+-influx into whole cells.
 camp-induced ca2+-influx in dictyostelium is controlled by at least two non-mitochondrial ca2+-stores: acidic stores and the endoplasmic reticulum. the acidic stores may comprise the contractile vacuole network, the endosomal compartment and acidocalcisomes. here the role of cv in respect to function as a potential ca2+-store was investigated.  dajumin-gfp labeled contractile vacuoles were purified 7-fold by anti-gfp-antibodies in a magnetic field. the purified cv were shown for the first time to accumulate and release ca2+. release of ca2+ was elicited by arachidonic acid or the calmodulin antagonist w the latter due to inhibition of the pump. the characteristics of ca2+-transport and ca2+-release of cv were compared to similarly purified vesicles of the er labeled by calnexin-gfp. since the cv proved to be a highly efficient ca2+-compartment we wanted to know whether or not it takes part in camp-induced ca2+-influx. we made use of the lvsa--mutant expected to display reduced ca2+-transport due to loss of calmodulin. we found a severe reduction of camp-induced ca2+-influx into whole cells.  the contractile vacuoles in dictyostelium represent a highly efficient acidic ca2+-store that is required for camp-induced ca2+-influx. 