©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence That the Pertussis Toxin-sensitive Trimeric GTP-binding Protein G Is Required for Agonist- and Store-activated Ca Inflow in Hepatocytes (*)

(Received for publication, July 10, 1995; and in revised form, August 14, 1995)

Leise A. Berven (1) Michael F. Crouch (2)(§) Frosa Katsis (3) Bruce E. Kemp (3) Lyn M. Harland (1) Greg J. Barritt (1)(¶)

From the  (1)Department of Medical Biochemistry, School of Medicine, Flinders University, G. P. O. Box 2100, Adelaide, South Australia 5001, the (2)Division of Neuroscience, John Curtin School of Medical Research, Australian National University, G. P. O. Box 334, Canberra, A. C. T. 2601, and the (3)St. Vincent's Institute of Medical Research, 41 Victoria Parade, Melbourne Victoria, 3065, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein alpha subunits, and carboxyl-terminal alpha-subunit synthetic peptides. An anti-G antibody and a G peptide (G Ile-Phe), but not a G peptide (G Ile-Phe), inhibited vasopressin- and thapsigargin-stimulated Ca inflow, had no effect on vasopressin-stimulated release of Ca from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca. An anti-G antibody also inhibited vasopressin-stimulated Ca inflow and partially inhibited vasopressin-induced release of Ca from intracellular stores. Immunofluorescence measurements showed that G is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, G was found principally at the cell periphery. It is concluded that the trimeric G-protein, G, is required for store-activated Ca inflow in hepatocytes and acts between the release of Ca from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca channel protein(s) in the plasma membrane.


INTRODUCTION

Receptor-activated calcium channels (RACCs) (^1)are present in most non-excitable and in some excitable animal cells and are responsible for allowing the inflow of Ca to specific regions of the cytoplasmic space and the refilling of intracellular Ca stores (most likely a region of the endoplasmic reticulum)(1, 2, 3) . For a number of cell types it has been shown that agonist-receptor complexes open at least two types of RACCs differing in selectivity for divalent cations(3) . The mechanism(s) by which RACCs are opened is poorly understood(1, 2, 3) . The hypothesis presently favored is the store-operated (capacitative) mechanism in which an increase in inositol 1,4,5-trisphosphate (InsP(3)) and the release of Ca from a region of the InsP(3)-sensitive store are prerequisites for channel activation(1, 2, 3) . This hypothesis is based, in part, on the observation that thapsigargin, which inhibits the endoplasmic reticulum (Ca + Mg)-ATPase causing the release of Ca from this organelle, leads to a stimulation of Ca inflow(2) . The results of a variety of experimental approaches have implicated the InsP(3) receptor(4) , a mobile intracellular messenger(5, 6, 7) , a monomeric G-protein(8, 9) , a trimeric G-protein (10, 11, 12, 13, 14) , protein phosphorylation(15) , and/or elements of the cytoskeleton (16) in the mechanism that couples the release of Ca from the endoplasmic reticulum to activation of the plasma membrane Ca channels.

In hepatocytes, agonists that employ RACCs include vasopressin, adrenaline, angiotensin II, and epidermal growth factor(11, 12, 13) . It has been shown previously that pretreatment of hepatocytes with pertussis toxin, or the microinjection of GDPbetaS, inhibits store-operated, as well as vasopressin-stimulated, Ca inflow. These treatments did not affect the release Ca from intracellular stores. This suggested that in addition to G, a slowly ADP-ribosylated pertussis toxin-sensitive trimeric G-protein is required for activation of the hepatocyte RACC(s)(11, 12, 13) . Since the two pertussis toxin-sensitive trimeric G-proteins present in hepatocytes at detectable levels are G and G(17, 18) , it was considered likely that one of these G-proteins is involved in activation of the hepatocyte RACC(s) (11, 12) . The aim of the present experiments was to identify the trimeric G-proteins involved in store-operated Ca inflow in hepatocytes. The approach employed used antibodies generated against peptides corresponding to a region of the carboxyl termini of G(19, 20, 21) and synthetic peptides corresponding to specific regions of the carboxyl termini of G(22, 23) . These antibodies and peptides have been shown by others to inhibit the activation of trimeric G-proteins(19, 20, 21, 22, 23) . Thapsigargin-stimulated Ca inflow is inhibited by an anti-G antibody and by a G peptide, indicating that the trimeric G-protein G controls store-operated Ca inflow in hepatocytes. Immunofluorescence experiments indicate that the G polypeptide is located in the interior of the hepatocyte and is not restricted in intracellular location to the plasma membrane.


EXPERIMENTAL PROCEDURES

Materials

A rabbit polyclonal anti-G antibody, which recognizes G, G, and G, was raised against the peptide KENLKDCGLF, a region of the carboxyl amino acid sequence for G, and a rabbit polyclonal anti-G antibody, which recognizes G and G, was raised against the peptide QLNLKEYNLV, a common region of the carboxyl amino acid sequence for G and G. The antibodies were prepared using peptides linked to keyhole limpet hemocyanin, affinity-purified as described previously(24) , and routinely stored at -70 °C in Tris-glycine buffer, pH 8.3. The abilities of the antibodies to recognize G (anti-G) and G, respectively, in extracts of hepatocytes were confirmed by Western blot analysis. The anti-G antibody detected a single band with an apparent molecular mass of 42 kDa and the anti-G antibody a single band with an apparent molecular mass of 43 kDa.

The peptides against which antibodies were raised, and the peptides IKNNLKDCGLF (G Ile-Phe (peptide G)) and IKNNLKECGLY (G Ile-Phe (peptide G)), corresponding to the carboxyl termini of G and G, respectively, were synthesized (as free COOH) by the Merrifield solid-phase synthesis procedure using an Applied Biosystems 430A synthesizer and were analyzed by quantitative amino acid analysis and mass spectrometry, as described previously(25) . Solutions of peptides G and G were prepared fresh each day by dissolving the peptide in a solution of 10 mM fura-2 in 125 mM KCl to give a concentration of 12 mM peptide in the microinjection pipette tip (estimated intracellular concentration 160 µM).

Fluorescein isothiocyanate (FITC)-conjugated rabbit IgG was obtained from Sigma-Aldrich, Castle Hill, New South Wales, Australia; and indocarbocyanine (Cy3) from Jackson, West Grove, PA. All other reagents were obtained from sources described previously(11) .

Microinjection of Fluorescent Dyes, Antibodies, and Peptides to Hepatocytes and Measurement of CaInflow

The isolation of hepatocytes, attachment of hepatocytes to coverslips coated with collagen, the microinjection of fura-2, antibodies, and peptides, and measurement of the fluorescence of single hepatocytes loaded with fura-2 were conducted as described previously(11) . The dilution factor for the microinjection of reagents to hepatocytes, determined previously(12) , was approximately 75-fold. Antibodies were concentrated in a buffer composed of 27 mM K(2)HPO(4), 8 mM Na(2)HPO(4), and 26 mM KH(2)PO(4) (adjusted to pH 7.3 by addition of KOH) (phosphate buffer) (26) using a Centricon-10 concentrator (Amicon Inc., Beverly, MA) at 4,000 times g for 30 min, stored at 4 °C, and used within 1-8 days. The final concentration of antibody in the microinjection pipette tip ranged from 2.5 to 3.0 mg/ml (estimated intracellular concentration 30-40 µg/ml). Antibodies were co-injected with fura-2 (10 mM in the pipette tip, estimated intracellular concentration 130 µM) at the concentrations indicated in the figure legends. In control experiments, the G antibody (2.2-3.0 mg/ml in the pipette tip) was mixed with blocking peptide KENLKDCGLF (1.7 mM in pipette tip) and co-injected with fura-2. Peptides G (Ile-Phe) and G (Ile-Phe) were dissolved in phosphate buffer, mixed with fura-2 to give 12 mM peptide and 10 mM fura-2 in the micropipette tip, and introduced to the cytoplasmic space of hepatocytes by microinjection. The fluorescence of FITC-conjugated rabbit IgG injected into hepatocytes was measured using excitation and emission wavelengths of 490 and 540 nm, respectively.

Immunofluorescence

Freshly isolated hepatocytes (10^6 cells/50-mm plastic Petri dish) were cultured for 24 h in William's Medium E, fixed with 4% (w/v) paraformaldehyde, and permeabilized using Triton X-100 as described by Lièvremont et al.(27) . After washing twice with 0.05% (w/v) Tween in PBS (Tween-PBS), the cells were incubated overnight with a given affinity-purified rabbit anti-G antibody (50 µg/ml) or with a given anti-G antibody which had been mixed with a molar excess of appropriate blocking peptide. The cells were washed five to six times with Tween-PBS to remove primary antibody and then incubated with donkey anti-rabbit IgG antibody conjugated to indocarbocyanine (Cy3) (1 in 200). Immunofluorescence was detected using a Nikon inverted microscope, a times 60 or times 100 oil immersion objective lens and a Bio-Rad MRC 1000 scanning confocal imaging system incorporating a krypton-argon laser.


RESULTS

Hepatocytes loaded with anti-G antibody exhibited a substantial inhibition of vasopressin-stimulated Ca inflow compared with control hepatocytes (Fig. 1a, solid line, cf.Fig. 1b). As reported previously(8, 11, 12, 19, 26) , there was some heterogeneity in the responses given by individual hepatocytes. Details of the total number of cells tested and the numbers of cells yielding a given type of response are set out in Table 1. In the majority of cells tested the ability of vasopressin to release Ca from intracellular stores was not affected by microinjection of the antibody (Table 1). Pretreatment of the anti-G antibody with the peptide against which the antibody was raised (the blocking peptide) prevented the inhibition of vasopressin-stimulated Ca inflow (Fig. 1a, broken line; Table 1). The anti-G antibody also inhibited thapsigargin-stimulated Ca inflow and caused some inhibition of thapsigargin-stimulated release of Ca from intracellular stores (Fig. 1c, Table 1). No inhibition of vasopressin-stimulated Ca inflow was observed in cells loaded with an anti-G antibody raised against a peptide which corresponds to a region near the amino terminus of G (results not shown).


Figure 1: Effects of anti-G and anti-G antibodies on vasopressin- and thapsigargin-stimulated Ca inflow. The fluorescence of single cells was measured as described under ``Experimental Procedures.'' Vasopressin (5 nM), thapsigargin (20 µM), and Ca (1.3 mM) were added at the beginning of the periods indicated by the horizontal bars. Anti-G antibody (estimated intracellular concentration 30-40 µg/ml) or anti-G antibody (estimated intracellular concentration 30 µg/ml) was co-injected with fura-2 (estimated intracellular concentration 130 µM). a and b, inhibition by anti-G antibody of vasopressin-stimulated Ca inflow. a, solid trace, anti-Gantibody; broken trace, anti-G antibody pretreated with blocking peptide; b, no antibody present. The solid and broken traces in a are representative of the results obtained for 1 of 14 cells (26 cells tested) and for 1 of 6 cells (8 cells tested), respectively (Table 1). The trace shown in b is representative of the results obtained for 1 of 21 cells (26 cells tested) (Table 1). c, inhibition by anti-G antibody of thapsigargin-stimulated Ca inflow. The solid (anti-G antibody present) and broken (no antibody) traces are representative of the results obtained for 1 of 8 cells (10 cells tested) and 1 of 8 cells (9 cells tested), respectively (Table 1). d, inhibition by anti-G antibody of vasopressin-stimulated Ca inflow. The solid (anti-G antibody present) and broken (control, no antibody present) traces are represenative of the results obtained for 1 of 5 cells (7 cells tested), and for 1 of 3 cells (3 cells tested), respectively (Table 1).





The effects of the anti-G antibody were compared with those of an antibody against G. This inhibited vasopressin-stimulated Ca inflow in most cells tested and caused partial inhibition of the vasopressin-stimulated release of Ca from intracellular stores (Fig. 1d, Table 1), as shown previously for hepatocytes by Yang et al.(19) . The possibility that the microinjected anti-G antibody was incompletely distributed in the cytoplasmic space was tested by microinjecting FITC-conjugated rabbit IgG to hepatocytes. The fluorescence signal diffused evenly in recipient cells within 5 min following the microinjection of the antibody, indicating that antibody microinjected to an hepatocyte is distributed throughout the cell within 5 min following its microinjection (results not shown).

The microinjection of peptide G (Ile-Phe) inhibited vasopressin-stimulated Ca inflow in almost all cells tested but had no effect on the ability of vasopressin to release Ca from intracellular stores (Fig. 2a, Table 1). By contrast, microinjection of peptide G (Ile-Phe), at the same concentration as that employed for peptide G, caused no inhibition of either Ca inflow or Ca release from intracellular stores induced by vasopressin (Fig. 2a, Table 1). Neither peptide caused an activation of Ca inflow in the absence of vasopressin or thapsigargin (results not shown). Thapsigargin-stimulated Ca inflow was also inhibited by peptide G. Complete inhibition was observed in 55% of the cells tested (Fig. 2b, cf.Fig. 2c, Table 1). Peptide G caused little or no inhibition of thapsigargin-stimulated Ca inflow (Fig. 2b, cf.Fig. 2c, Table 1). However, both peptides caused some inhibition of thapsigargin-induced release of Ca from intracellular stores (Fig. 2b, cf.Fig. 2c, Table 1). In 7 out of 11 cells (peptide G) and in 3 out of the 4 cells (peptide G) the inhibition of the thapsigargin-induced release of Ca from intracellular stores by the peptide was associated with an inhibition of thapsigargin-induced Ca inflow. This suggests there may be some correlation between the effects of the peptides on the release of Ca from intracellular stores and their effects on Ca inflow.


Figure 2: The effects of G Ile-Phe and G Ile-Phe peptides on vasopressin- and thapsigargin-stimulated Ca inflow. The fluorescence of single cells was measured as described under ``Experimental Procedures.'' Vasopressin (5 nM), thapsigargin (10 µM), and Ca (1.3 mM) were added at the beginning of the periods indicated by horizontal bars. When present, G (Ile-Phe) or G (Ile-Phe) (estimated intracellular concentration 150 µM) was co-injected with fura-2. a, effects of G and G peptides on vasopressin-stimulated Ca inflow. The solid (G peptide present) and broken (G peptide present) traces are representative of the results obtained for 1 of 7 cells (18 cells tested) and for 1 of 9 cells (10 cells tested), respectively (Table 1). b and c, effects of G and G peptides on thapsigargin-stimulated Ca inflow. The solid (G peptide present) and broken (G peptide present) traces in b are representative of the results obtained for 1 of 11 cells (20 cells tested) and for 1 of 8 cells (12 cells tested), respectively (Table 1). The trace shown in c (control, no peptide present) is representative of the results obtained for 1 of 11 cells (15 cells tested) (Table 1).



Since the results of the experiments conducted with anti-G antibodies and site-specific G peptides suggest that G is required for the activation of Ca inflow, the intracellular location of G was investigated by immunofluorescence, using anti-G antibody as the primary antibody and anti-rabbit IgG antibody coupled to the fluorescent dye Cy3. In most cells, immunofluorescence, which was dependent on anti-G antibody, was found to be distributed throughout the cytoplasmic space as well as in most parts of the cell periphery (Fig. 3a). This distribution is seen more clearly at higher magnification (Fig. 3b). The fluorescence signal given by anti-G antibody was not observed when the anti-G antibody was omitted or when this antibody was pretreated with the blocking peptide (results not shown). A pattern of immunofluorescence similar to that given by anti-G antibody was observed when an anti-G antibody was employed as the primary antibody (results not shown). In contrast to the results obtained with the anti-G antibody, when anti-G antibody was employed, the fluorescence signal was largely confined to the periphery of the cell, adjacent to the plasma membrane (Fig. 3, c and d). The fluorescence given by anti-G antibody was abolished when anti-G antibody was omitted or when this antibody was pretreated with blocking peptide (results not shown). The cells labeled with the anti-G antibody exhibited a much more defined location of immunofluorescence at the cell periphery than that exhibited by cells labeled with the anti-G antibody (Fig. 3c, cf.Fig. 3a). These results indicate that while G is distributed in the plasma membrane and in various regions of the cytoplasmic space, G is located predominantly at the plasma membrane.


Figure 3: Intracellular localization of G and G in hepatocytes using immunofluorescence. Fluorescence images of cells treated with anti-G antibody (a and b) and anti-G antibody (c and d). Freshly isolated hepatocytes were cultured for 24 h, fixed, permeabilized, treated with the indicated primary rabbit anti-G polyclonal antibody and a goat anti-rabbit IgG secondary antibody conjugated to Cy3, and the fluorescence viewed by scanning confocal fluorescence microscopy, using a times 60 (a, c) and times 100 (b, d) objective lens, as described under ``Experimental Procedures.'' The images are from a scan 1 µm in depth in a plane located approximately half way between the bottom and top of the cell. The scale bars represent 10 µm. The results shown are those obtained for a representative sample of the cells observed in one of five experiments which gave similar results.




DISCUSSION

Previous studies with hepatocytes which utilized pertussis toxin, GTPS, and GDPbetaS have shown that vasopressin-dependent Ca inflow requires a pertussis toxin-sensitive trimeric G-protein in addition to the pertussis toxin-insensitive G-protein G, which is required for the activation of phospholipase Cbeta and the subsequent generation of InsP(3)(11, 12, 13) . The conclusion that vasopressin- and thapsigargin-stimulated Ca inflow requires the action of G and/or G is consistent with the observation reported here that the anti-G antibody, but not the anti-G antibody treated with blocking peptide, inhibited vasopressin- and thapsigargin-stimulated Ca inflow. Moreover, the observation that, when present at the same concentration as peptide G, peptide G did not inhibit vasopressin- and thapsigargin-stimulated Ca inflow indicates that the observed inhibitory effects of peptide G are most likely due to inhibition of the action of G. Taken in conjunction with the observations that the only detectable pertussis toxin-sensitive trimeric G-proteins in hepatocytes are G and G(16, 17) , the results reported here indicate that the pertussis toxin-sensitive trimeric G-protein required for activation of the hepatocyte plasma membrane receptor-activated Ca channel is G.

The conclusion that G is required for store-activated Ca inflow in hepatocytes is consistent with the observation that G is distributed in regions of the cytoplasmic space as well as at the plasma membrane, in contrast to G which was found at the plasma membrane of the hepatocyte. ADP-ribosylation of G catalyzed by pertussis toxin is very slow (11, 19) , consistent with a location of some G in the cytoplasmic space.

The partial inhibition of the thapsigargin-induced release of Ca from intracellular stores by the anti-G antibody (which would not be expected to bind to other intracellular proteins) and the G and G peptides was unexpected especially in view of the absence of an inhibition of vasopressin-stimulated Ca release. This preferential inhibition of thapsigargin-induced Ca release may reflect some form of steric interaction between G and the thapsigargin-sensitive (Ca + Mg)-ATPase proteins.

The observations that (a) the anti-G antibody completely inhibited vasopressin-stimulated Ca inflow (present results) and (b) the only known action of G is to activate phosphoinositide-specific phospholipase Cbeta (28) provide further evidence which indicates that an increase in InsP(3) is a necessary prerequisite for vasopressin activation of Ca inflow in hepatocytes (cf. the conclusion reached previously on the basis of studies with GDPbetaS and heparin(11) ). The observation that the anti-G antibody completely inhibits thapsigargin-stimulated Ca inflow as well as vasopressin-stimulated Ca inflow also provides further evidence that the process of store-operated Ca inflow is a necessary part of the mechanism of activation of the plasma membrane Ca channel by vasopressin in hepatocytes (cf. the conclusion reached previously on the basis of results with pertussis toxin which was also shown to block both vasopressin- and thapsigargin-stimulated Ca inflow(12, 13) ).

The failure of the anti-G antibody to completely inhibit vasopressin-induced release of Ca from intracellular stores may be due to a failure of the injected antibody to bind to all G molecules, possibly because the affinity of the anti-G antibody for G is low or G is in an environment that restricts the accesses of the antibody. Others have also reported that, relative to the effects of anti-G antibodies, longer incubation times are required in order to detect the inhibition by anti-G antibodies of phospholipase C in intact cells(19, 20) . Another possible explanation for incomplete inhibition by the anti-G antibody of vasopressin-induced release of Ca is that, in hepatocytes, there is a species of phosphoinositide-specific phospholipase C which can be activated by seven transmembrane-spanning receptors through a mechanism which does not involve G(28) . However, no evidence for such a pathway in hepatocytes has so far been reported. Furthermore, G is unlikely to be involved in the activation of phosphoinositide-specific phospholipase Cbeta in hepatocytes, since first, this function of G in hepatocytes has not been reported, and second, the anti-G antibody and the G peptide caused no inhibition of vasopressin-stimulated release of Ca from intracellular stores. It is noteworthy that in several cells the anti-G antibody inhibited vasopressin-stimulated Ca inflow with little effect on the release of Ca from intracellular stores. One possible explanation for this observation is that only a small region of the intracellular Ca stores (most likely the endoplasmic reticulum near the plasma membrane) is involved in activation of the plasma membrane Ca channels.

Based on the results obtained, the sequence of events emerging for the activation by vasopressin of RACCs in hepatocytes is likely to include the following steps: formation of the vasopressin-receptor complex, activation of G, activation of phospholipase C, an increase in InsP(3) at the periphery of the cell, release of Ca from the endoplasmic reticulum in this region, activation of G, and activation of one or more RACCs. Since the anti-G antibody and the G peptide inhibit thapsigargin-stimulated Ca inflow (in the absence of added vasopressin and hence in the absence of the formation of the vasopressin-receptor complex) activation of G would not involve its interaction with a seven-transmembrane-spanning receptor protein. One possible role of G may be to regulate the movement of Ca between components of the endoplasmic reticulum (8, 29, 30) or interaction of the endoplasmic reticulum with the plasma membrane.

The proposed role of G in store-activated Ca inflow in hepatocytes does not exclude a role for a low molecular weight G-protein, as proposed for mast and mouse lacrimal acinar cells, in part, on the basis of the observation that GTPS inhibits store-activated Ca inflow in these cell types(8, 9) . Indeed, other studies conducted in this laboratory have also shown that a relatively high concentration of GTPS inhibits thapsigargin-stimulated Ca inflow in hepatocytes. (^2)Furthermore, the action of G may be complimentary to that of a Ca influx factor (5, 6, 7) .


FOOTNOTES

*
This work was supported by a grant from the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Wellcome Senior Research Fellowship in Medical Science in Australia.

To whom correspondence should be addressed: Dept. of Medical Biochemistry, School of Medicine, Flinders University, G. P. O. Box 2100, Adelaide, South Australia, 5001, Australia. Tel.: 61-8-2044260; Fax: 61-8-3740139; mbgjb@cc.flinders.edu.au.

(^1)
The abbreviations used are: RACC, receptor-activated calcium channel; G-protein, GTP-binding regulatory protein; InsP(3), inositol 1,4,5-trisphosphate; Ca, intracellular free Ca concentration; Ca, extracellular Ca concentration; PBS, phosphate-buffered saline; peptide G (Ile-Phe), Ile-Lys-Asn-Asn-Leu-Lys-Asp-Cys-Gly-Leu-Phe; peptide G (Ile-Phe), Ile-Lys-Asn-Asn-Leu-Lys-Glu-Cys-Gly-Leu-Tyr; FITC, fluorescein isothiocyanate; GDPbetaS, guanyl-5`-yl thiophosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate.

(^2)
L. A. Berven and G. J. Barritt, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge Kekulu Fernando for the preparation of hepatocytes; Diana Tanevski and Jennie McCulloch, who prepared the typescript; Professor Ian Gibbins, Department of Anatomy and Histology, for advice on the immunofluorescence experiments; Dr. Reinhold Penner, Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, Gottingen, Germany, for drawing the authors' attention to the idea of G-protein-regulated transfer of Ca between intracellular organelles; and Dr. Peter Kolesik, Confocal Facility, The University of Adelaide, South Australia, for performing the confocal microscopy.


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