©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Regulation of Ca Release-activated Ca Influx by Heterotrimeric G Proteins (*)

(Received for publication, July 11, 1995; and in revised form, September 12, 1995)

Xin Xu (1)(§) Kenichiro Kitamura(§) (2) Kim S. Lau (1) Shmuel Muallem (1)(¶) R. Tyler Miller (2)

From the  (1)Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the (2)Department of Medicine and Pharmacology, University of Florida, Gainesville, Florida 32610

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The least understood aspect of the agonist-induced Ca signal is the activation and regulation of the Ca release-activated Ca influx (CRAC) across the plasma membrane. To explore the possible role of heterotrimeric G proteins in the various regulatory mechanisms of CRAC, continuous renal epithelial cell lines stably expressing alpha and the constitutively active alpha were isolated and used to measure CRAC activity by the Mn quench technique. Release of intracellular Ca by agonist stimulation or thapsigargin was required for activation of CRAC in all cells. Although the size of the internal stores was similar in all cells, CRAC was 2-3-fold higher in alpha- and alpha-expressing cells. However, the channel was differentially regulated in the two cell types. Incubation at low [Ca], inhibition of the NOS pathway, or inhibition of tyrosine kinases inhibited CRAC activity in alpha but not alpha cells. Treatment with okadaic acid prevented inhibition of the channel by low [Ca] and the protein kinase inhibitors in alpha cells. These results suggest that expression of alpha dominantly activates CRAC by stabilizing a phosphorylated state, whereas expression of alpha makes CRAC activation completely dependent on phosphorylation by several kinases. G proteins may also modulate CRAC activity independently of the phosphorylation/dephosphorylation state of the pathway to increase maximal CRAC activity. Furthermore, our results suggest a general mechanism for regulation of CRAC that depends on coupling of receptors to specific G proteins.


INTRODUCTION

The [Ca] signal evoked by G protein-dependent Ca mobilizing agonists such as bradykinin (BK) (^1)and carbachol involves activation of inositol 1,4,5-trisphosphate-mediated Ca release from internal stores (IS), which is followed by activation of a plasma membrane located Ca influx pathway to increase [Ca]. Subsequently IS and plasma membrane Ca pumps remove Ca from the cytosol restoring [Ca] to basal levels(1, 2) . Periodic repetition of this sequence results in [Ca] oscillations(1, 2) .

A poorly understood aspect of the Ca signal is the nature and regulation of the Ca release-activated Ca influx (CRAC) pathway, although significant advances have been made in recent years(1, 3) . Ca release from IS is required and sufficient for activation of Ca entry(3, 4) . Ca release initiated by agonist stimulation or by inhibition of IS Ca pumps activates the same CRAC pathway(3) . Patch clamp experiments in several cell types(5, 6, 7, 8) have identified a Ca release-activated Ca current (I) which can also be followed by measurement of Mn influx through I and quenching of Fura 2 fluorescence(8, 9, 10) .

Multiple regulatory mechanisms of I have been reported. CRAC and I appear to be sensitive to [Ca](11, 12, 13) , where high [Ca] rapidly and partially inactivates the influx(11, 13) . In multiple cell types, CRAC is inhibited by tyrosine kinase inhibitors, suggesting that tyrosine kinase-dependent phosphorylation regulates I(14, 15, 16, 17) . In basophils (18) and lacrimal acinar cells (19) a GTP-dependent and GTPS-inhibitable mediator is required for channel activation. In pancreatic acini (20, 21, 22, 23) intestinal cells (24) and macrophages, (25) cGMP modulates CRAC activity.

The mechanism linking Ca release from IS and activation of I is not well defined. When the channel is regulated by cGMP, Ca release from IS activates a nitric-oxide synthase (NOS) to generate NO, increase cGMP and stimulate Ca influx(22, 23) . However, the channel is not activated by cGMP without depletion of IS Ca(22) . Recently, much attention has been given to activation of I by a diffusible messenger(s). In Xenopus oocytes (26) and basophils(18) , sustained activation of I requires a cytosolic factor that can be stabilized by inhibition of protein phosphatases with okadaic acid (OA)(26) . A small, P(i)-containing molecule, named Ca influx factor, was extracted from Jurkat T cells and was suggested to be the diffusible messenger responsible for activation of CRAC(27) . Subsequent studies in oocytes further support (28, 29) and challenge (30, 31) the suggestion that Ca influx factor is the universal activator of Ca influx required for reloading of IS upon termination of cell stimulation.

The family of heterotrimeric G proteins couples receptor-initiated events in the plasma membrane to effector molecules inside the cell. The alpha subunits contain the primary information that determines the specificity of receptor and effector interactions. Galpha(q) is widely expressed in mammalian tissues and regulates phospholipase C beta isoforms, thereby regulating inositol 1,4,5-trisphosphate production and Ca release from IS. The functions of the alpha chains of the alpha/alpha family are only beginning to be understood. alpha is widely expressed in mammalian tissues(32, 33) . Expression of alpha increases Na/H exchange activity(34, 35, 36) , transforms cells in tissue culture, and increases expression of immediate early genes(37, 38, 39) . More recently we showed that stable overexpression of Galpha in the continuous MCT renal epithelial cell line increases expression and activity of the inducible form of NOS through a transcriptional mechanism. (^2)

Because of the central role of G proteins in Ca signaling we used MCT cells stably expressing alpha and the activated mutant alpha to study the role of G proteins in regulating CRAC activity. We report here that expression of alpha and alpha markedly increase CRAC influx (2-3-fold) initiated by agonist stimulation or depletion of IS with Tg. In cells expressing alpha CRAC activation required elevated [Ca], the activity of protein kinases and could be stably activated by OA, an inhibitor of protein phosphatases. In alpha-expressing cells, depletion of IS from Ca maximally activated CRAC independent of [Ca] and cellular kinases/phosphatase activities. These findings suggest that (a) G proteins modulate CRAC influx by direct and indirect interactions and (b) CRAC influx is regulated by multiple kinase/phosphatase-dependent events.


MATERIALS AND METHODS

Fura 2 was purchased from Molecular Probes; tissue culture medium, serum, and G-418 were from Life Technologies, Inc. [^3H]Arginine was from Amersham Corp. Tissue culture plasticware was from Falcon. Other chemicals were from Sigma or, if molecular biology grade, from Fisher.

Cell Culture

MCT cells, an SV40-transformed mouse proximal tubule cell line were a gift from Eric Neilson(41) . Cultures were maintained in Dulbecco's modified Eagles' medium/Ham's F-12 (50:50) plus 5% fetal bovine serum. Experiments were performed when cells were approximately 90% confluent. Serum and G-418 were removed 18-24 h before experiments.

Construction of cDNAs and Expression in Mammalian Cells

The cDNA constructs and vectors for stable expression of alpha and alpha were described previously(34, 42) . The cDNAs were expressed in MCT cells using pZem-228 that contains a mouse metallothionein promoter. All studies were performed without heavy metal induction of the alpha chains because there was an adequate expression of the alpha subunits without induction. A clonal cell line expressing alpha and neomycin-resistant pooled cells expressing alpha were used for studies. Pooled neomycin-resistant MCT cells expressing pZem-228 without an insert were used as controls (Neo cells)(34) .

Measurement of NOS Activity

NOS activity was measured in whole cells as conversion of [^3H]arginine to [^3H]citrulline. Cells in 24-well cluster trays were washed once with phosphate-buffered saline and then incubated with 400 µl of phosphate-buffered saline (pH 7.4) containing 4.2 mM KCl, 1 mM CaCl(2), 7.8 mM glucose, and 1 µCi of [^3H]arginine, and incubated at 37 °C for 15 min. At time 0, BK, L-NAME, or vehicle were added and incubation was continued for additional 10 min at 37 °C. The reactions were stopped with 5 M trichloroacetic acid, and the supernatants were extracted with ether and neutralized with 25 mM HEPES. Extracts were added to Dowex columns (AG8 200-400-mesh) and eluted with buffer containing 20 mM HEPES (pH 5.5), 2 mM EDTA, and 2 mM EGTA, and counted. Each sample was corrected for recovery by the addition of approximately 1200 cpm of [^14C]citrulline to the stop solution. The counts in each well were normalized for protein and are expressed as cpm/mg of protein/min. Each point represents the mean of triplicate determinations.

Measurement of [Ca](i) and Mn Influx

Cells were released from culture plates by a brief incubation with trypsin/EDTA and washed twice with solution A. The composition of solution A was (in mM): 140 NaCl, 5 KCl, 1 MgCl(2), 1.5 CaCl(2), 10 glucose, 10 HEPES (pH 7.4 with NaOH), and 0.1% bovine serum albumin. The cells were resuspended in solution A and incubated with 5 µM Fura 2/AM for 20 min at 37 °C. After one wash with solution A, the cells were resuspended and kept on ice until use. [Ca](i) and Mn influx were measured either in cell suspension or by image acquisition and analysis. For measurement in cell suspension (most presented results), samples of Fura 2-loaded cells (50 µl) were added to 1.45 ml of a warm (37 °C) solution A and fluorescence was recorded at excitation wavelengths of 340 and 360 nm and at an emission wavelength of 500 nm. After various incubations Mn influx was initiated by adding 0.25 mM Mn to the cuvette. When the cells were incubated in Ca-free medium, Mn influx was initiated by adding a mixture of Ca and Mn to final concentrations of 1.5 and 0.25 mM, respectively. When the rate of Mn influx was measured, fluorescence quenching by Mn precluded calibration of the signals to calculate [Ca](i). In this case [Ca](i) changes were evaluated from control experiments under identical conditions, except that the fluorescence signal was calibrated and Mn influx was not measured. Calibration of the signal was as described (22) . For single cell measurements, the Fura 2-loaded cells were plated on glass coverslips that formed the bottom of a perfusion chamber. The image acquisition and analysis setup was described in detail in previous publications(43) .


RESULTS

CRAC Activity in Neo-, alpha-, and alpha-expressing MCT Cells

CRAC was measured by the Mn quench technique. The activity of CRAC was estimated from the initial rate of Fura 2 fluorescence quench. Fig. 1shows that stimulation of Neo control MCT cells with a maximal concentration of BK increased Mn influx by about 3-fold (3.19 ± 0.35, n = 7), whereas complete depletion of the IS of Ca with Tg resulted in about a 6-fold (5.93 ± 0.48, n = 15) increase in the rate of influx.


Figure 1: Rate of CRAC influx in Neo control cells and cells expressing alpha and alpha. Cells loaded with Fura 2 and incubated in solution A were stimulated with 50 nM BK or exposed to 0.1 µM Tg. When [Ca] was stabilized, Mn influx was initiated by addition of 0.25 mM Mn to the medium. After about 2 min the cells were lysed by addition of 50 µM digitonin (Dig.) to obtain the 100% quench at each condition. The effect of digitonin on fluorescence of BK-stimulated cell was deleted for clarity. Each experiment shown represents at least 8 similar observations with cells from different subpassages.



Stable expression of the G protein alpha subunits alpha and the GTPase-deficient alpha(q) (alpha) dramatically increased CRAC influx in BK or Tg-stimulated cells. The basal rate of Mn influx was similar in all cells, although it tended to be slightly higher in alpha cells (17 ± 8%, n = 8 relative to paired Neo control or alpha cells). After stimulation of the pooled alpha-expressing cells with BK, CRAC activity was 6.78-fold higher than that measured in alpha controls. Treatment of alpha cells with Tg was more effective than BK stimulation in increasing CRAC activity. Tg increased CRAC activity by 16.05-fold above control, which was about 2.49-fold higher than BK in the same cells and 3.01 ± 0.08-fold higher (n = 9) than in Tg-treated Neo control cells. In cells from the clone expressing alpha, BK and Tg stimulation caused similar increases in CRAC activity, which was 10.14- and 11.25-fold above control alpha, respectively. These rates were between 3.18 ± 0.27-fold (n = 4) (BK) and 1.9 ± 0.17-fold (n = 9) (Tg) above the rates measured in similarly treated Neo controls. Similar results were observed in three additional alpha clones and in three clones expressing wild type alpha(q), although in all cases expression of alpha increased CRAC activity more than expression of alpha(q). In the case of alpha, recent experiments showed that expression of the GTPase-deficient alpha increased Ca and Mn influx rates similar to alpha (data not shown).

Since CRAC activity increases with increased Ca depletion of IS(3, 44, 45, 46) , a possible explanation for the increased activity of CRAC in alpha- and alpha-expressing cells is an increased IS pool size and depletion during Tg treatment. This was not the case since measurement of pool size by exposing cells in Ca-free medium to high concentrations of ionomycin (47) produced similar increases in [Ca](i) in all cell types. Thus, 5 µM ionomycin increased [Ca](i) of cells incubated in albumin- and Ca-free medium to 848 ± 66 (Neo), 861 ± 58 (alpha), and 797 ± 55 (alpha) in 3-5 experiments from different subpassages. In addition, Tg caused similar [Ca](i) increase in all cells (see below).

Regulation of CRAC Activity by [Ca](i)

To study the role of [Ca](i) in CRAC activation we measured CRAC activity during Ca depletion and [Ca](i) restoration. Fig. 2shows the activity of CRAC at different [Ca](i) during Tg treatment of Neo, alpha, and alpha cells. Tg treatment in Ca-free medium caused a similar increase in [Ca](i) in all cell types, demonstrating the similar size of the IS Ca pool in all cells. After 4 min of incubation with Tg to maximally deplete the pool and reduce [Ca](i) to resting levels (point a in all experiments) CRAC was almost maximal in alpha cells, intermediate in Neo cells and minimal in alpha cells.


Figure 2: Dependence of CRAC activity on [Ca]. Neo, alpha, or alpha cells were incubated in Ca-free solution A (for experiments a-c in each case) or solution A containing 1.5 mM CaCl(2) (trace d in each case) and stimulated with 0.1 µM Tg. At different times after exposure to Tg (a and d, 4 min; b, 30 s) cells were exposed to a mixture of Ca and Mn (a and b) or Mn (d) to measure the rate of Mn quench. Cells incubated in Ca-free medium and Tg for about 4.5 min were exposed to 1.5 mM CaCl(2) for about 30 s to increase [Ca](trace c) before exposure to Mn for measurement of fluorescence quench rate.



The time courses of CRAC activation during IS Ca depletion and during restoration of [Ca](i) for the three cell types were measured using the protocol of Fig. 2and are illustrated in Fig. 3. In alpha cells, activation of CRAC required Ca release from IS, but the sustained CRAC activity did not require a sustained elevation of [Ca](i). CRAC activity was near maximal after 3 min of incubation with Tg, and remained active when [Ca](i) was reduced to resting levels (Fig. 3, panel A). CRAC activity did not change with a subsequent increase in [Ca](i) to about 350 nM (Fig. 3, panelB). In contrast, in cells expressing alpha, activation of CRAC required Ca depletion and persistently high [Ca](i). During the Ca depletion protocol, CRAC activity mirrored [Ca](i), increasing during the first min of incubation with Tg and returning to the basal rate as [Ca](i) was reduced. Increasing [Ca](i) then reactivated CRAC (Fig. 3, panel B, open circles). CRAC activation was time-dependent, requiring a 2-min incubation at 37 °C, although [Ca](i) was maximal 20-25 s after addition of [Ca] (Fig. 2). The requirement for [Ca](i) to activate CRAC in the Neo control cells was intermediate between that of the cells expressing alpha and the cells expressing alpha. However, CRAC activity was 2-3-fold higher in the cells expressing the G proteins than in the Neo cells (Fig. 3, panel B). The requirement of high [Ca](i) for activation of CRAC in alpha cells is likely to be independent of NOS, since these cells show high NOS activity in the unstimulated state (see below). Hence expression of alpha increased CRAC activity and stabilized it in a [Ca](i)-dependent form and expression of alpha increased CRAC activity and stabilized it in a [Ca](i)-independent form.


Figure 3: Time course of CRAC activation during Ca depletion (A) and [Ca] restoration (B). The protocol of figure 2 was used to measure rates of Ca influx during Ca depletion and [Ca] restoration. 100% control was taken as the rate of quench in Fig. 2(trace d) of alpha cells. At different times during Tg treatment in Ca-free medium (25 s, 1, 2, 3, or 5 min) of the different cell types a mixture of Ca and Mn was added to measure the rate of fluorescence quench (panel A). Then 1.5 mM Ca was added to increase [Ca] as in Fig. 2(trace c) and Mn quench rates were measured 0.5, 1, 2 or 5 min after addition of Ca (panel B). The results in the figure are the mean of 3 (Neo), 5 (alpha), and 4 (alpha) experiments. S.E. were between 5 and 12% of the signals and are not shown for clarity.



Regulation of CRAC Activity by the NO Pathway

CRAC activity can be regulated by cGMP generation through the activation of NOS(22, 23) . In previous studies we used a molecular and biochemical analysis to demonstrate the transcriptional induction of NOS by alpha to increase NOS protein and activity in extracts of MCT cells.^2Fig. 4shows the activity of NOS in intact MCT cells under resting and stimulated conditions. Basal NOS activity was about 8-fold higher in cells overexpressing alpha than in control Neo cells. Expression of alpha also increased basal NOS activity but to a much lower extent than in cells expressing alpha. Similar results were obtained when NO(2) production was measured (data not shown). Importantly, BK stimulation increased NOS activity in Neo controls and in cells expressing alpha. The effect of BK on NOS activity in alpha-overexpressing cells was less prominent, probably because of the high basal activity, which reduced the signal/noise ratio. L-NAME blocked the majority of citrulline production from arginine, indicating that citrulline was produced by NOS. Hence these cells should provide an excellent system to study the role of NOS in regulating the Ca influx pathway.


Figure 4: NOS activity in intact MCT cells. Cells were preincubated for 15 min in arginine-deficient medium and [^3H]arginine. They were then stimulated with 50 nM BK in the presence or absence of 5 mML-NAME for an additional 10 min. The reactions were stopped with trichloroacetic acid, extracted with ether, and [^3H]citrulline separated from [^3H]arginine by column chromatography. Each value is the mean of triplicate samples ± S.E. The experiment shown is representative of three others with similar results.



Fig. 5depicts representative tracings, whereas Fig. 6summarizes the results of multiple experiments. Treatment of control, alpha, or alpha cells with the arginine analog L-NAME, which inhibits NOS activity(48) , or NO(2), an NO donor(22, 49) , had no effect on CRAC activity when IS were loaded with [Ca](i) (Fig. 6). However, L-NAME almost abolished CRAC activity in alpha cells, whether it was activated by BK (Fig. 6) or Tg ( Fig. 5and Fig. 6). Inhibition of CRAC by L-NAME was completely reversed by bypassing NOS inhibition with NO(2) to supply the cells with NO and activate the soluble guanylyl cyclase. cGMP was as effective as NO(2) in reversing the inhibitory effect of L-NAME (data not shown).


Figure 5: Effect of NOS inhibition and a NO donor on CRAC activity in cells expressing alpha and alpha. Cells in solution A were treated for 10 min at 37 °C with or without 5 mML-NAME. Samples of the cells were also incubated for 2 min with 15 mM NO(2) before stimulation with 0.1 µM Tg. After 4 min of exposure to Tg, fluorescence quench measurements were initiated by addition of Mn to the incubation medium.




Figure 6: Inhibition of Ca influx by L-NAME and reversal by NO(2) in cells expressing alpha but not alpha. The procedure used to treat the cells and measure fluorescence quench was the same as that described in the legend to Fig. 5. The various cell types were stimulated with 50 nM BK or 0.1 µM Tg as indicated. The rate of fluorescence quench in Tg-treated alpha cells was taken as 100% control. The figure shows the mean ± S.E. of 3-4 experiments.



In contrast to the finding in alpha cells, L-NAME and NO(2) had no effect on BK or Tg-activated CRAC activity in alpha cells. Thus, L-NAME did not inhibit CRAC activity in BK- or Tg-treated cells and increasing cGMP with NO(2) did not increase CRAC activity beyond that measured in the absence of NO(2). Again, in Neo controls both NO-sensitive and NO-insensitive components of CRAC activity (a mixture of the behavior observed in alpha- and alpha-overexpressing cells) was found, with lower maximal CRAC activity.

Effect of Protein-tyrosine Kinase Inhibitors

Protein-tyrosine kinase (PTK) inhibitors reduce the activity of CRAC(14, 15, 16, 17) . Fig. 7shows the effect of the most commonly used PTK inhibitor, genistein on CRAC activity in the three cell types. Pretreatment of alpha-overexpressing cells with genistein strongly inhibited CRAC activity, with an IC of between 2-3 µM and maximal inhibition at 10 µM. At this concentration genistein had minimal effect on CRAC activity in Neo cells, but inhibition was observed at higher concentrations, similar to those reported in other cell types(16, 17) . At concentrations up to 50 µM, genistein had no effect on CRAC activity in alpha-overexpressing cells. Hence, overexpression of alpha made CRAC particularly sensitive to PTK inhibitors, whereas overexpression of alpha prevented inhibition of CRAC by PTK inhibitors.


Figure 7: Effect of genistein on CRAC activity. Cells were incubated with the indicated concentration of genistein or daidzein for 5 min at 37 °C before stimulation with 50 nM BK (data not shown) or 0.1 µM Tg. After 4 min of incubation in the presence of Tg, Mn was added to measure the rate of Fura 2 fluorescence quench. The upper panel shows the traces recorded from Neo cells treated with 50 µM genistein or 50 µM daidzein and alpha cells treated with 10 µM genistein or 10 µM daidzein. Daidzein up to 100 µM had no effect in either cell type. The lower panel shows the mean ± S.E. of 4 (alpha and Neo) or 5 (alpha) experiments of cells treated with Tg. The 100% control in each case was taken as the rate of quench measured in Tg-treated cells in the absence of drugs.



Role of Phosphorylation/Dephosphorylation

CRAC activity can be increased by inhibition of protein phosphatases with OA (26, 50) . Therefore, we tested the effect of OA treatment on CRAC activity in the three cell types. Fig. 8shows representative tracings while Fig. 9summarizes the results of 2-4 experiments of selective conditions. Treatment of all cells with OA for 10 min at 37 °C had no effect on resting [Ca](i), but significantly increased the rate of Mn influx. In resting alpha cells OA increased Mn influx by about 1.5-fold and had a small additive effect on Tg-treated alpha cells. Similar results were obtained in alpha cells (Fig. 9). Treatment of Neo controls with OA also increased CRAC activity, but activity never approached that in cells expressing alpha or alpha.


Figure 8: Effect of okadaic acid on CRAC activity in cells expressing alpha. alpha-overexpressing cells were treated with 0.1 µM OA or 0.1 µM of the nonactive analog 1-nor-okadaone for 5 min at 37 °C before addition of 10 µM genistein. After an additional 5-min incubation at 37 °C, treated and untreated cells were stimulated with 0.1 µM Tg for 4 min before addition of Mn to measure the rate of fluorescence quench.




Figure 9: Okadaic acid reverses CRAC inhibition by low [Ca], NOS, and PTK inhibitors. Cells were treated with 0.1 µM OA, 5 mML-NAME, or 10 µM genistein as indicated in the figure and then stimulated with 0.1 µM Tg. After stabilization of [Ca], fluorescence quench was measured by addition of Mn to the incubation medium. Samples of cells were also maintained in Ca-free medium during treatment with OA and stimulation with Tg to measure the effect of OA on CRAC activity of alpha cells at low [Ca] as detailed in Fig. 2, time point a. The figure shows the averages of 3 (Neo), 2 (alpha), or 4 (alpha) experiments. Please compare the effect of low [Ca], L-NAME and genistein in this figure to those in Fig. 2(middle panel), 6, and 7.



The most important finding of this set of experiments is that treatment with OA completely prevented the inhibition of CRAC activity by low [Ca](i) (Fig. 9), L-NAME (Fig. 9), and genistein ( Fig. 8and Fig. 9) in alpha-overexpressing cells. OA prevented the inhibition of CRAC by low [Ca](i) and the kinase inhibitors whether CRAC was activated by Tg treatment (Fig. 9, middle columns) or BK (data not shown). The nonactive analogue of OA, 1-nor-okadaone, had no effect ( Fig. 8for Tg and genistein).


DISCUSSION

The activation and regulation of the CRAC pathway have not been characterized in molecular terms. Recent studies suggest that Ca influx factor, a small P(i)-containing molecule (27) that is sensitive to cellular phosphatases(50) , may be the ubiquitous activator of CRAC. This suggestion has been both challenged (30, 31) and supported (28, 29) by studies using oocytes and lacrimal acinar cells. In a number of experimental systems CRAC is regulated by the NO metabolic pathway (20, 21, 22, 23, 24, 25) , GTP-dependent and GTPS-inhibited processes (18, 19) , protein-tyrosine kinases(14, 15, 16, 17) , and cytochrome P-450 metabolites(51, 52) . No individual regulatory mechanism can account for all aspects of CRAC activity in all cases, and it is possible that several mechanisms may be operative in one cell type(3, 17, 24) . A common feature of the CRAC regulatory mechanisms described so far appears to be involvement of a phosphorylation/dephosphorylation mechanism. Indeed, inhibition of protein phosphatases with OA prolongs and augments CRAC activity(26, 50) .

In the present study we used a new approach, that of overexpressing specific G protein alpha subunits to study the regulation of CRAC. Our results suggest that G proteins can regulate CRAC activity, both by influencing the phosphorylation/dephosphorylation state of the system and by a phosphorylation-independent mechanism. These two modes are illustrated in Fig. 10. Below we discuss the evidence in favor of such regulatory effects.


Figure 10: A model summarizing the effect of heterotrimeric G proteins on CRAC activity.



Release of Ca from the IS of Neo control cells by BK stimulation or treatment with Tg increased CRAC activity. Tg was more effective than BK in activating CRAC because it mobilized all the Ca present in IS, whereas BK mobilized about 60% of the stored Ca. This was the case whether Ca was measured in cell populations or at the single cell level. A similar situation existed in alpha-overexpressing cells. In alpha-expressing cells BK and Tg were equally effective in mobilizing IS Ca and activating CRAC. A close relationship between Ca mobilization and Ca influx has been reported previously in several cell types(3, 44, 45, 46) . Our studies extend these findings to show that even when CRAC activity is up-regulated its regulation by Ca content of the IS is maintained. An additional point of these experiments is that overexpression of the alpha subunits did not alter the fundamental features of Ca signaling. The cells must have adjusted to overexpression of alpha, and in particular the constitutively activated alpha, to prevent persistent activation of the Ca signaling system. This adjustment probably represents activation of the normal ``turn off'' mechanism for signals initiated by alpha(q) or alpha.

Neo control cells express a number of G protein alpha chains including alpha(q) and alpha(34) . In these cells CRAC activity has two components. The first requires persistently high [Ca](i) and is sensitive to inhibition by inhibitors of NOS and protein-tyrosine kinases. The second component is relatively independent of [Ca](i) and is not sensitive to inhibition by these agents. Expression of specific heterotrimeric G protein alpha subunits stabilizes CRAC activity in one of these forms. Overexpression of alpha increased total CRAC activity and stabilized the form that requires persistently high [Ca](i) and is sensitive to inhibition by L-NAME and genistein. A 2-min incubation at high [Ca](i) was required for maximal activation of CRAC in alpha cells (Fig. 3B). This [Ca](i) dependence of CRAC activation is not likely to reflect NOS activation since during agonist stimulation NOS is maximally activated within 30 s (22, 23) and alpha cells show high NOS activity also in the unstimulated state (Fig. 4). High [Ca](i) may influence CRAC activity through activation of a [Ca](i)-dependent protein kinase(s), since the requirement for [Ca](i) can be bypassed by OA. Such will be a new mechanism of CRAC regulation. Inhibition of a phosphatase by alpha was required to unmask this mechanism. Expression of alpha also made CRAC activity completely dependent on an active NOS pathway. Finally CRAC activity in alpha-overexpressing cells was completely blocked by genistein at a concentration at least 5-fold lower than that reported for other systems(15, 16, 17) . When taken together these observations suggest that alpha stabilizes the CRAC pathway in a general inactive state.

Overexpression of alpha also increased total CRAC activity, but stabilized the second, [Ca](i)-independent, L-NAME- and genistein-insensitive form of the pathway. Activation of CRAC still required Ca release from IS, but [Ca](i) could be reduced to resting levels (Fig. 2) without inactivating CRAC (Fig. 3). Inhibition of the NO pathway ( Fig. 5and Fig. 6) or protein-tyrosine kinases (Fig. 7) did not inhibit CRAC activity. Thus, once CRAC was activated by Ca release from IS of alpha-expressing cells, it could not be inactivated by reducing [Ca](i) or inhibiting protein kinase activities.

The simplest interpretation of these observations is that expression of alpha stabilizes a dephosphorylated state of CRAC to make CRAC activity completely dependent on protein kinases, whereas expression of alpha constitutively activates CRAC by stabilizing a phosphorylated state of the channel itself or an accessory regulatory protein (Fig. 10). Such an interpretation is supported by the effect of the protein phosphatase inhibitor OA, which caused CRAC activity in alpha cells to behave much like that in alpha cells. These findings can help explain previous observations concerning regulation of CRAC activity. In pancreatic acini(20, 21, 22, 23) , intestinal cells(24) , and macrophages(25) , but not other cells(3) , CRAC activity is regulated by cGMP through the NO pathway. Similarly, in some (14, 15, 16, 17) but not necessarily all (3) cells, CRAC activity is inhibited by protein kinase inhibitors. It is possible that the relative expression and/or influence of alpha and alpha(q) on CRAC activity determines the dominant regulatory mode of CRAC in a given cell type.

Expression of alpha or alpha increases CRAC activity by a mechanism beyond affecting the phosphorylation state of the CRAC pathway (Fig. 10). The maximal activity of CRAC in alpha and alpha cells was approximately 3-fold higher than that in Neo controls or cells overexpressing other wild type or constitutively active alpha subunits(40) . We could not find a condition, including long incubation at high [Ca](i) of Tg-treated cells, increased cGMP by a NO donor, or treatment with OA of control cells or cells exposed to Tg and cGMP, which augmented CRAC activity in Neo cells to the level seen in the cells that overexpress alpha and alpha. Furthermore, the effect of alpha and alpha was specific to these alpha subunits in that similar overexpression of alpha(s) reduced CRAC activity in the same cell type(40) . Augmentation of CRAC activity did not require activated alpha subunits. The properties of the [Ca](i) signal in alpha(q)- and alpha-expressing cells was similar. Recently we isolated MCT cells overexpressing alpha^2 and found that their [Ca](i) signal resembled that of alpha-overexpressing cells. Thus, alpha(q) and alpha appear to up-regulate CRAC activity beyond affecting the phosphorylation state of the pathway. It is not clear at present whether expression of alpha and alpha(q) increased the number of CRAC channels or the activity of existing channels.

In summary, by expressing different G protein alpha subunits such as alpha(i), alpha(s)(40) , and alpha and alpha (present study) we were able to show that CRAC activity can exist in a dephosphorylated state (alpha overexpression) or phosphorylated state (alpha overexpression) that modulates the activity of the pathway. This may explain the diversity of CRAC activity and its regulation observed in different cell types.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 39839. 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.

§
These two authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-2593; Fax: 214-648-8685.

(^1)
The abbreviations used are: BK, bradykinin; CRAC, Ca release-activated Ca influx; IS, intracellular stores; NOS, nitric oxide synthase; NO, nitric oxide; OA, okadaic acid; Tg, thapsigargin; L-NAME, N-nitro-L-arginine; PTK, protein-tyrosine kinase; GTPS, guanosine 5`-3-O-(thio)triphosphate.

(^2)
K. Kitamura, W. D. Singer, R. A. Star, S. Muallem, and R. T. Miller, submitted for publication.


ACKNOWLEDGEMENTS

We thank Paul McLeroy for technical assistance and Mary Vaughn for expert administrative assistance.


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