Jan 15, 2016 - tent activator of the guanine nucleotide-binding pro- tein G., specifically stimulates a sustained inward whole cell flux of calcium through a novel ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 267,No. 2, Issue of January 15,)p: 883+8,1992 rinted in U.S.A.
The Guanine Nucleotide-bindingProtein GsActivates a Novel Calcium Transporter in Xenopus Oocytes* (Received for publication, August 20, 1991)
Philip M. Murphy$ andDavid McDermotte From the Laboratory of Host Defenses. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
Calcium influx is an important aspect of receptorcytoplasmic calcium concentration is achieved by energy remediated signal transduction, yet limited information quiring pumps. is available regarding the pathways of calcium influx Several G proteins have been implicated in the regulation into nonexcitable cells. We showthattreatmentof of ion channels (3-5, 13, 14).In patchclamp studies, G,a has oocytes from Xenopus laevis with cholera toxin, a po- been shown to directly activate cardiac and skeletal muscle tent activator of the guanine nucleotide-binding pro- L-type voltage-dependent calcium channels by a membranetein G., specificallystimulatesasustainedinward delimited process that is independent of cytoplasmic second whole cell flux of calcium through a novel membrane messengers (3-5). In thepresent paper, we describe the proptransporter. The calciumis distributed into a mobiliz- erties and regulation of a distinct G,-activated calcium transable pool. Theflux is voltage-independent and is com- porter expressed by the oocyte of Xenopus laevis. pletely and specifically blocked by microinjection of oocytes with an antiserum directed against G.a. The EXPERIMENTAL PROCEDURES flux is not activated by treatment of the cells with Materials-Cholera holotoxin, B-oligomer, and pertussis toxin forskolin or 8-bromo-cyclic adenosine monophosphate were azide-free and were from List (Campbell, CA). A23187, nifediindicatingthatthe effect of Gsa onthetransporter pine, diltiazem, verapamil, and forskolin were from Calbiochem. All occursindependently of adenylylcyclaseactivation. salts and ATPyS’were from Sigma. Alldivalent and trivalentcations Transporter activity is insensitive to benzyl amiloride, were used as the chloride salt. Benzyl amiloride was from Research does not require a sodium gradient, andis not stimu- Biochemicals (Natick, MA). Female laboratory-bred X. laeuis were lated by external calcium, indicating that it is not a purchased from Nasco, Fort Atkinson, WI and were maintained as sodium-calciumexchanger.TheGs-activated flux is previously described (15). Affinity-purified antisera directed against the carboxyl-terminal decapeptides of the a subunits of G. (anti-RM) dramatically potentiated by lanthanum ion and other and Go (anti-QI),as well as synthetic RM and QI peptides were gifts trivalent cations but not by any ofsix divalent cations from A. Spiegel and P. Goldsmith (National Institutes of Health.) that were tested; all other known calcium channels and Calcium Flux Assays-Oocyteswere obtained, maintained, and exchangers are, in contrast, potently blocked bylan- microinjected as previously described (15). Defolliculated oocytes thanum. Thedivalent cationcadmium inhibited trans- were incubated in “standard medium” (96 mM NaCI, 1 mM MgCL, porter activity in a concentration-dependent manner. 1.8 mM CaC12, 2 mM KCl, pH 7.45) with or without cholera toxin for h at 20 “C. To measure calcium uptake, oocytes were then transThis novel calcium transporter may be important for 24 ferred to “uptake medium” (calcium-free standard medium, with or receptor-mediatedcalciuminfluxintheoocyteand without fresh toxin, to which 50 pCi/ml 45Ca2+(Du Pont-New Engperhaps othercell types. land Nuclear, specific activity 25 mCi/mg) was added). Uptake me-
The concentration of ionized calcium in the cytoplasm of cells from higher organisms is typically 10,000-fold less than that in the extracellular milieu. This gradient serves as a powerful battery whose discharge through a variety of membrane transportersis linked to theactivation state of the cell. Calcium has been shown to enter the cell by selective channels whose open probabilities can be regulated by membrane potential, ligand, G protein, or second messengers such as inositol trisphosphate, cyclic AMP, and calcium itself (1-10). Calcium entry also occurs by coupled exchange with sodium ions (11, 12). The precise mechanisms that regulate calcium entry may differ in different cell types. Restoration of the low
dium contained 44 p~ 45Caz+ and approximately 1p M nonradioactive Ca2+.All other test substances were added to the extracellular fluid a t the beginning of the incubation in ‘%a2+. In the microinjection experiments, peptides and antisera were directly introduced into the oocyte cytoplasm 1 h before exposure to extracellular toxin. Oocytes were microinjected with synthetic peptides alone or with antisera that had been incubated with peptide a t room temperature for 20 min (preabsorbed antisera). The design of the [3SS]methionineuptake experiments was identical to that described for calcium with the exception of the tracer molecule. After incubation for the indicated duration in uptake medium, oocytes were washed 10 times over 10 minutes with standard medium and homogenized individually with 100 &I of 1%sodium dodecyl sulfate. 45Ca2+uptake was then determined by counting the lysates in 10 ml of scintillation mixture in a Hewlett-Packard scintillation counter. For calcium efflux experiments, oocytes were first loaded by incubation in uptake medium for 3 h. They were then washed with isotope-free uptake medium. Calcium efflux was determined by incubating the oocytes for 24 h in 100 pl of isotope-free uptake medium with or without 2 pg/ml cholera toxin. The medium and thelysed oocyte werethen counted. Data are presented as the mean rt standard error of the mean of replicate determinations and derive from experiments performed with oocytes from four separate frogs. Receptor-mediated Calcium Efflux Assay-Human phagocytic cells
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: The Laboratory of Host Defenses, Bldg. 10, Rm. llN113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-2877; Fax: 301-402-0789. The abbreviation used is: ATPrS, adenosine 5’-0-(thiotriphos§ Howard Hughes Medical Institute-National Institutes of Health phate). Research Scholar.
‘
883
Transporter G,-activated Calcium
884
express a P2 nucleotide receptor which is activated by ATP and is linked to calcium mobilization (15). Poly(A)’ RNA waspurified from HL60 neutrophils (HL60 cells stimulated for 48 h with dibutyryl cyclic AMP) as described (15). Oocytes weremicroinjected with RNA samples in a total volume of 50 nl/oocyte 1-2 days after harvesting and were then incubated at 18-20 “C for at least 3 days in medium. Oocytes were then incubated with or without 2 pg/ml cholera toxin for 24 h and were then loaded with 45Ca2’and washed as described above. Individual oocyteswere stimulated with 100 pM ATPyS in individual wells of a 96-well tissue culture plate containing 100 p1 of medium. Three 100-pl samples were collected and analyzed by liquid scintillation counting: ( a ) the final 100-pl wash of 20-min duration, before application of ligand; ( b ) fluid containing the stimulus, removed after a 20-min incubation with the oocyte; and (c) the oocyte solubilized in 1%sodium dodecylsulfate after the 20-min stimulation. Acquired ligand-dependent calcium efflux wascalculated as the total "Cas+ that was released and as the percentage of loaded 45Ca2+that was released in response to stimulation with ligand, defined as ( b a ) and ( ( b - a)+(b c ) ) X 100, respectively. Data are presented as the mean & standard error of the mean of replicate determinations. Membrane Potential Measurements-Oocytes were impaled with a single glass electrode filled with 3 M KC1 with a resistance of 5-15 megohms. Transmembrane potentialwas measured with reference to an electrode placed in the bath using an Axoclamp IIA amplifier (Axon, Burlingame, CA).
+
RESULTS
Cholera Toxin Stimulates the Rate of CalciumInflux by Oocytes-The heat-stable toxin of Vibrio cholerae has a variety of biological effects which are due to its ability to ADPribosylate the CY subunit of G, (16-18). When Xenopus oocytes were incubated in cholera toxin, the rate of uptake of 45Ca2+ was stimulated 2-10-fold as compared with oocytes incubated without cholera toxin (Fig. 1).The flux was linear over a 3-h uptake period with 1 min as the earliest time point. In the experiment shown in Fig. 1,calcium uptake by oocytes treated with cholera toxin was 5 fmol/min/oocyte, a &fold increase over the untreated controls. Pertussistoxin, which ADPribosylates the Gi but not the G, class of G proteins, had no effect (Fig. 2 A ) . Incubation for at least 2 h with cholera toxin was required before the effect on calcium flux could be detected (not shown). This lag period, which is required for other effects of cholera toxin, is due to the time required for the toxin to be internalized and reach its target after binding to plasma membrane GM1 gangliosides (19). Calcium uptake by the oocyte was affected by cholera toxin in a concentration-dependent manner (Fig. 2B). The threshold concentration, 0.1 ng/ml, is consistent with the known potency of cholera toxin with respect to its other biological effects (20). No effect of cholera toxin on calcium efflux was observed. Thus, increased calcium uptake was due to stimu-
Cholera toxin
0
20
40
60
lncubatlon Time (mln)
FIG. 1. Cholera toxin stimulates the rate of calcium uptake by Xenopus oocytes. Oocytes were preincubated in the presence (closed circles) or absence (open circles) of 2 pg/ml cholera toxin for 24 h. “Cas+ uptake over time was then measured. Data derive from 10 oocytes/condition and are representative of two separate experiments.
.
N T F T C T
.
, 42
. -10
. d
. d
Log [Cholera Toxin] (g/ml)
FIG. 2. Cholera toxin specifically stimulates calcium uptake by Xenopus oocytes in a concentration-dependent manner. “Cas+ uptake over a 3-h period was measured as described under “Experimental Procedures.” A: NT, CT,and PT refer to oocytes that were preincubated for 24 h with no toxin, 2 pg/ml cholera toxin, or 2 pg/ml pertussis toxin, respectively. Data are pooled from six separate experiments. The S.E. was less than 10% of the mean. B, oocytes were preincubated with the indicated concentration of cholera toxin for 24 h prior to and during the uptake assay. Data derive from at least 5 oocytes/condition.
lation of influx and not inhibition of efflux (not shown). That the toxin does not permeabilize the cell ina nonspecific manner was shown by an identical uptake assay with [35S] methionine as the tracer solute (not shown). In contrast to its effect on calcium uptake, amino acid uptake was reduced by 50% in cells treated with cholera toxin. The cholera toxin effect is also observed when toxin is microinjected into the oocyte, suggesting that it is acting on an intracellulardeterminant. Cholera toxin can beboiled without loss of its ability to stimulate calcium influx, consistent with its reported thermal stability (21). The B oligomer, which is important for holotoxin internalization but lacks ADP-ribosyl transferase activity (19), does not stimulate the calcium flux. The magnitude of the effect is greater than that observed over an identical time period in oocytes stimulated with 1 W M calcium ionophore A23187 (Table I). Distribution of Calcium into a Mobilizable Pool-A human phagocyte P2 nucleotide receptor that is known to couple to a pertussis toxin-sensitive G protein and calcium mobilization was expressed in the oocyte by microinjection of poly(A)+ RNA from HL60 neutrophils (15). Acquired ATPyS-dependent 45Ca2+efflux was measured from oocytes incubated with and without cholera toxin. Base-line 45Ca2+efflux was low, indicating that the cell-associated calcium was not freely diffusible. With ATP-yS stimulation, however, 45Ca2+efflux .was greatly increased from cholera toxin-treated oocytes as compared with control oocytes, indicating that theincreased cell-associated calcium was stored in a mobilizable pool (Fig. 3). The percentage of total loaded 45Ca2+that was released into themedium in response to stimulation with ATPyS was not affected by cholera toxin treatment; that is, no effect of cholera toxin on the receptor-mediated signal transduction pathway was detected. This contrasts with the inhibitory effect of pertussis toxin on P2 receptor signal transduction (15). T h e Cholera Toxin Effect Is Mediatedby G,-That the transporter is activated by G,was suspected because of the known mechanism by which cholera toxin exertsits biological effects (16, 17). To test thishypothesis, the following affinity purified antisera were used in microinjection experiments. Anti-RM is directed against a synthetic peptide that corresponds exactly to the carboxyl-terminal 10 residues of the CY subunit of G, as predicted from the cDNA sequences of higher species (22) including X . laeuis (23). Anti-QI is directed againstasynthetic peptide that corresponds exactly to a divergent carboxyl-terminal 10 residues of the CY subunit of
Transporter
Calcium
TABLEI Mechunism of cholera toxinstimulatwn of calcium influx in Xenopus oocytes Results of six separate experiments are shown (A-F). Oocytes were incubated in the presence or absence of 2 pg/ml cholera toxin for 24 h prior to incubation with the indicated substance in uptakemedium. In experiment C, oocytes were microinjected with 50 nl of water, 2 pg/ml cholera toxin, or 2 pg/ml B-oligomer and then incubated in the absence of extracellular additive for 24 h. Calcium uptake by individual oocytes wasmeasured and is reported as the percent of the mean value of control cells. Control cells were those injected with water in C, and those incubated in the absence of toxin in all other experiments. Data derive from 9-10 oocytes/condition. The standard error of the mean was less than 10% in all cases. Addition
"Ca" uptake
% of control
Expt. A B-oligomer (2 pg/ml) Cholera toxin Expt. B Boiled cholera toxin (2 pg/ml)" Cholera toxin Expt. C B-Oligomer microinjected Cholera toxin microinjected Cholera toxin in bath Expt. D
106 220 257 243
120 780 1040 140
A23187 (1p M )
8-bromo-cyclicAMP ( 1 mM) 67 200 Cholera toxin Expt. E Forskolin (10 p ~ ) 59 Cholera toxin 223 228 Forskolin (10 p M ) cholera toxin Expt. F Benzyl amiloride (400 pM) 75 Cholera toxin 247 Benzyl amiloride (400 p ~ +)cholera 189 toxin a Cholera toxin was boiled for 10 min prior to addition to cells.
+
Ligand:
-
+ NT
885
G,-activated
-
uptake by control oocytes (not incubated with cholera toxin). However, microinjection of oocytes with anti-RM inhibited the cholera toxin-stimulated inward flux of calcium in a concentration-dependent manner (Fig. 4). One hundred percent inhibition was achieved by injecting 50 nl of antiserum at 0.5 pg/pl prior to treatmentof the cells with cholera toxin. When oocytes were injected with 0.5 pg/pl (approximately 200 nM) anti-RM antiserum that had been preabsorbed with 100 p~ RM peptide, cholera toxin-stimulatedcalcium uptake was restored to 82 +- 9% (n = 8) of the value measured in water-injected control cells (p = 0.10). In contrast, control peptide was unable to absorb the inhibitory effect of antiRM. The specificity of the inhibitory effect of anti-RM was further demonstrated by control experiments with anti-QI antiserum, the G,a-specific antiserum. Microinjection of oocytes with anti-QI did not inhibitthe cholera toxin-stimulated calcium flux. Instead, a concentration-dependent stimulation was observed (see below). Anti-QI had no effect on calcium flux in cells that were not treated with cholera toxin. These data strongly indicate that thecholera toxin effect is mediated by G,a. Assuming an antibody molecular mass of 150,000 daltons and an oocyte volume of 1 pl, the ICs0 for inhibition by anti-RM was 16 nM. This value agrees with that reported for inhibition of isoproterenol-stimulated adenylylcyclase activity in membranes from S49 lymphoma cells, an activation pathway that is known to be stimulated by G , (22, 24-26). A major functional role of G,is to activate adenylylcyclase, resulting inthe accumulation of cyclic AMP in thecytoplasm (24). Treatment of cells with forskolin, which directly activates adenylylcyclase, or with 8-bromo-cyclic AMP, which elevates the intracellular cyclic AMP concentration did not increase calcium uptake by the oocyte indicating that the cholera toxin effect was mediated by G , through amechanism that is independent of adenylylcyclase activation (Table I). Properties of the Calcium Transporter-The stimulated inward flux of calcium could be due to activation of a calcium channel or an exchanger. Benzyl amiloride, an inhibitor of Na+-Ca2+exchange activity ( l l ) , had little effect on cholera toxin stimulation of calcium influx (Table I). Moreover, addition of 1.8 mM unlabeled Ca2+to the medium led to inhibition of 45Ca2+influx (Fig. 5 ) . Stimulation should occur if a
d
+ CT
FIG. 3. The cholera toxin-activated calcium transporter imports calcium to a mobilizable pool. Oocytes were microinjected with 50 ng of HL60 neutrophil poly(A)+RNA. Three days later cells were incubated for an additional 24 h instandard medium containing no toxin (NT)or 2 pg/ml cholera toxin (CT).Oocyteswere then loaded with "Ca" as described under "Experimental Procedures." Calcium mobilization by the oocyte was then assessed by measuring 4sCa2+efflux over 20 min in response to standard medium alone (-) or medium containing 100 p M ATPyS (+). Mobilization of calcium by the acquired P2 nucleotide receptor was complete by 20 min after ligand application. Data derive from 5 oocytes/condition and are representative of three separate experiments.
Drosophila Go.' The antiserawere of similar titer anddid not cross-react by immunoblot analysis. Microinjection with either antiserum had no effect on the basal rate of calcium P. Goldsmith and A. M. Spiegel, unpublished data.
3" 9"
0'1 0.0
'
'
0.1
0.2
0.3
0.4
0.5
1
[Antlbody] Kgglrnl
FIG.4. Cholera toxin-stimulated 4sCaz+influx by Xenopus oocytes is mediated by G.a. Oocytes were microinjected with 50 nl of the indicated concentration of anti-QI antiserum, specific for Goa (open circles) or anti-RM antiserum, specific for G.a (closed circles). Oocytes were then incubated with 2 pg/ml cholera toxin for 24 h, at which point 45Ca2+ uptake was measured. The mean uptake of "Ca2+ by water-injected cells incubated in the absence of cholera toxin was 740 & 76 cpm ( n = 9) and is indicated by the broken horizontal line. Injection of either antiserumin the absence of cholera toxin treatment had no effect on the rate of basal calcium uptake by the cells. Data derive from 10 oocytes/concentration and are representative of three independent experiments.
886
G,-activated Calcium Transporter of a sodium-calcium exchanger due to theremoval of external sodium ions (12). In contrast, when cholera toxin-treated oocytes were depolarized, an additional calcium flux appeared that was not inhibited but instead was dramatically potentiated by lanthanum ion. DISCUSSION
The magnitude of the G,-activated calcium flux which we have described is sufficient to exert important changes on the calcium content and distribution in the oocyte. During a 3-h O 10 20 30 40 50 incubation in 45Ca2+, 0.5 pmol is typically imported by each 1 incubation Time in %a" (hrs) p1 oocyte. If this were distributed homogeneously in the free FIG.5. Competition by external Ca2+ with GB-stimulated ionized form, an intracellular concentration of 0.5 p M 45Ca2' 4aCaz+influx. Oocytes were incubated in standard medium with 2 would be achieved. Since the calculated extracellular concenpg/ml cholera toxin for 24 h. Oocytes were then incubated in uptake tration of 45Ca2+ , during the uptake assay is only 44 p ~ much medium (closed circles)or standard medium supplemented with 45Ca2' greater effects of G. activation onoocyte calcium homeostasis (open circles) for the indicated duration. Note that uptake medium would be anticipated in media containing concentrations of and standard medium contained 1p~ and 1.8 mM unlabeled calcium ion, respectively, whereas both media contained the same concentra- ionized calcium in the physiologic range (1-4 mM). Although this calcium transporter is clearly activated by tion of "Ca2+ (44 p M ) . Data derive from 10 oocytes/condition. G,, the enhanced uptake of calcium by oocytes that were microinjected with anti-QI antiserum prior to treatmentwith A cholera toxin (Fig. 4) suggests that Go may actually inhibit the activity of the transporter. This is consistent with the reported ability of Go to inhibit neuronal voltage-dependent Cholera toxln calcium channels (6, 14). Adenylylcyclase is another example \ of an effector molecule that can be oppositely regulated by two distinct G proteins,G, and Gi. Although a plasma membrane channel is probably responsible for the flux that we have described, the properties of the lncubatlon Time (min) putative channel, particularlythe ability of lanthanum ion to FIG.6. Lanthanum ion potentiates the G.-activated calcium potentiate itsactivity, areclearly not consistent with those of transporter activity: concentration dependence and time voltage-dependent calcium channels expressed by excitable course. A, concentration dependence. Oocytes were preincubated in cells (1).The receptor-operated calcium channel described by standard medium in the presence (closed circles) or absence (open Benham and Tsien (2) is also distinct by virtue of its insencircles) of 2 pg/ml cholera toxin for 24 h. %a2 '+ uptake was then measured over 3 h in uptake medium containing the indicated con- sitivity to cadmium ion. The FMLP-activated cation channel centration of lanthanum ion. In the absence of lanthanum, cholera in neutrophils is opened by astimulus-dependent rise in toxin stimulated calcium uptake 2-fold. Data derive from at least 9 [Ca2+Iiand is not thought to involve G, (12). oocytes/condition and are representative of six independent experiBiological membranes are not permeable to lanthanum ion ments. B, time course. After preincubation for 24 h in the presence (28, 29) so that its ability to markedly potentiate the cholera (closedcircles) or absence (open circles) of 2 pg/ml cholera toxin, 4aCa2+ uptake was measured over time in uptake medium containing toxin effect is most likely due to interaction of lanthanum with an extracellular site of a plasma membrane transporter. lanthanum ion 100 pM. It is evident that charge and valency, and not atomic radius, Na+-Ca2+exchanger were mediating the effect (11). Cholera are the principal determinants of the potentiating activity, toxin (2 pg/ml) did not significantly effect the oocyte mem- since cerium and chromium ions were also active but six were inactive. The lanthanumeffect brane potential. Moreover, voltage-dependent calcium chan- different divalent cations the concentranel blockers such as verapamil, nifedipine, and diltiazem did was strikingly concentration-dependent. When tion exceeded 100 p ~ lanthanum , activity was lost. not inhibit the cholera toxin-stimulated flux (not shown). To account for these observations, we speculate that the Lanthanum ion has been reported to potently inhibit all known calcium transporters (1,11).Lanthanum by itself had calcium transporter contains a contact sitefor G,a as well as little effect on calcium uptake by the oocyte over a concentra- two allosteric sites that bind lanthanum with different affintion range from 1 p M to 1 mM. However, over a narrow ities. Interaction of the transporter with G,a could mediate , poten- transporter activation by converting a channel to an open concentration range peaking at 100 p ~ lanthanum tiated the cholera toxin effect by 25-50-fold (Fig. 6). Other conformation, for example. Binding of lanthanum ion to the trivalent cations such as cerium and chromium, but none of high affinity site could potentiate transporter activation by six divalent cations, were able to potentiate thecholera toxin stabilizing the open conformation. Binding of a second lanactivity (Fig. 7A). In particular, the divalent cation cadmium thanum ion to the low affinity site could cause an additional inhibited the cholera toxin-stimulated flux (Fig. 7 B ) . Cad- allosteric conformational change and mediate transporter inmium sensitivity is also a propertyof sodium-calcium exchan- activation. Additional effects of cholera toxin on transmembrane ion gers and certain voltage-dependent calcium channels (1, 11). Fig. 8 directly demonstrates the uniqueness of the oocyte flux have been described which are opposite to those we have G,-activated calcium transporter. In thisstudy, oocytes were found in the oocyte. In rat basophilic leukemia cells a 2-3fully depolarized by incubation in 96 mM KC1. This maneuver fold increase in the initial rate of 45Ca2+influx was observed activated a calcium flux that was completely inhibited by only in cells stimulated with antigen (30, 31). In contrast, lanthanum ion. This result is compatible with the activation cholera toxininhibited the antigen receptor-mediated inof endogenous oocyte voltage-dependent calcium channels crease in [Ca*+li in the human T-cell line Jurkat (32, 33). (27) by means of the voltage step and/or with the activation While these effects may be mediated by G,, another cholera
A
G,-activated Calcium Transporter
887
FIG. 7. Effect of divalent and trivalent cations on the G.-activated calcium transporter. Oocytes were preincubated in standard medium with (open bars)or without (closed bars) 2pg/ ml cholera toxin for 24 h. Calcium uptake was then measured over 3 h in uptake medium containing the indicated cation 100 p M (A) or containing the indicatedconcentration of cadmium ion (B). Data derive from at least 9 oocytes/ condition.
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4
-5
.4
-3
Log [Cd2‘] (M)
2. Benham, C. D., and Tsien, R. W. (1987) Nature 328,275-278 3. Mattera, R., Graziano, M. P., Yatani, A., Zhou, Z., Graf, R., Codina, J., Birnbaumer, L., Gilman, A. G., and Brown, A. M. (1989) Science 243,804-807 4. Yatani, A., Codina, J., Imoto, Y., Reeves, J. P., Birnbaumer, L., and Brown, A. M. (1987) Science 2 3 8 , 1288-1292 5. Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895 6. Ewald, D. A., Pang, I.-H., Sternweis, P. C., and Miller, R. J. (1989) Neuron 2, 1185-1193 7. Toselli, M., Lang, J., Costa, T., and Lux, H. D. (1989) Pflugers Cholera toxln: + + “ + + Arch. Eur. J. Physiol. 415, 255-261 La” 1ool”: + + + + 8. Kuno, M., and Gardner, P. (1987) Nature 326,301-304 9. Gross, R. A,, and MacDonald, R. L. (1989) J. Neurophysiol. 61, NaCl KC1 97-105 FIG. 8. Effect of membrane depolarization on the lan- 10. von Tscharner, V., Prod’hom, B., Baggiolini, M., and Reuter, H. thanum-potentiated, G.-activated calcium transporter. 00(1986) Nature 324, 369-372 cytes were preincubated in standard medium for 24 h in the presence 11. Simchowitz, L., and Cragoe, E. J., Jr. (1988) Am. J.Physiol. 254, or absence of 2 pg/ml cholera toxin as indicated below the n axis. C150-Cl64 4sCa2+uptake was then measured over 3 h in uptake medium (NaC1) 12. Nicoll, D. A,, Longoni, S., and Philipson, K. D. (1990) Science or in uptake medium where NaCl was replaced with 96 mM KC1 250,562-565 (KCl), in the presence (closed bars) or absence (open burs) of lan- 13. Yatani, A., Codina, J., Brown, A. M., and Birnbaumer, L. (1987) thanum ion 100 p~ as indicated below the z axis. Data derive from Science 235,207-210 10 oocytes/condition. 14. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wittig, B. (1991) Nature 353, 43-48 toxin-sensitive G protein could be involved (34-37). Finally, 15. Murphy, P. M., and Tiffany, H. L. (1990) J. Biol.Chem. 265, 11615-11621 cholera toxin has been shown to function as a calcium iono(1990) in ADP-Ribosylnting Toxins and G-proteins (Moss, phore in jejunal brush border vesicles (38) and to create ion 16. Ui,J.,M.and Vaughan, M., eds) pp. 45-47, American Society of channels in artificial planar membranes that incorporate the Microbiology, Washington, D. C. GM1 ganglioside binding site for the toxin (39-41). The vari- 17. Freissmuth, M., and Gilman, A.G. (1989) J. Biol.Chem. 264, ability of the effects of cholera toxin on cellular calcium 21907-21913 homeostasis may reflect the richness of distinct calcium trans- 18. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science 252,802-808 porters that areexpressed by different cell types. In summary, we have described the properties of a novel 19. Van Heyningen, S. (1977) Biol. Reu. 52, 509-549 inward calcium transporter expressed by the Xenopus oocyte 20. Middlebrook, J. L., and Dorland, R. B. (1984) Microbiol. Reu. 48, 199-220 that is activated byG,, potentiated by lanthanum ion, and 21. Gill, D. M. (1976) Biochemistry 15,1242-1252 inhibited by cadmium ion and that is independent of voltage 22. Simonds, W. F., Goldsmith, P. K., Woodard, C. J., Unson, C. G., and adenylylcyclase. The ability of this transporter to be and Spiegel, A. M. (1989) FEBS Lett. 249, 189-194 activated by G, independently of adenylylcyclase activation is 23. Olate, J., Martinez, S., Purcell, P., Jorquera, H., Codina, J., Birnbaumer, L., and Allende, J. E. (1990) FEBS Lett. 268,27similar to the effect of G, on L-type voltage-dependent cal31 cium channels (3-5). Together these data indicate that G, and, by extension, G,-coupled receptors may independently 24. Kaslow, H. R., Johnson, G. L., Brothers, V. M., and Bourne, H. R. (1980) J. Biol. Chem. 255, 3736-3741 activate at least two distinct effector systems, adenylylcyclase 25. Graziano, M. P., Casey, P. J., and Gilman, A. G. (1987) J. Biol. and membrane calcium transporters in both excitable and Chem. 262,11375-11381 non-excitable cells. The transporter that we have described 26. Graziano, M. P., Freissmuth, F., and Gilman, A.G. (1989) J . may be an important mediator of calcium influx in the XenBiol. Chem. 264,409-416 opus oocyte as well as in othercell types. 27. Lester, H. A., Snutch, T. P., Leonard, J. P., Nargeot, J., Dascal, N., Curtis, B. M., and Davidson, N. (1989) Ann. N. Y. Acad. Acknowledgments-We thank A. Spiegel and P. Goldsmithfor Sci. 560,174-182 antisera andE. Gallin for performing the oocyte membrane potential 28. Boucek, M. M., and Snyderman, R. (1976) Science 193,905-906 measurements. We thank E. Gallin, J. I. Gallin, and H. Malech for 29. Kwan, C.-Y., and Putney, J. W., Jr. (1990) J. Bid. Chem. 265, critical reading of the manuscript. 678-683 30. Narasimhan, V., Holowka, D., Fewtrell, C., and Baird, B. (1988) REFERENCES J. Bid. Chem. 263, 19626-19632 31. McCloskey, M. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,72601. Tsien, R. W., Hess, P., McCleskey, E. W., and Rosenberg, R. L. (1987) Annu. Reu. Biophys. Biophys. Chem. 16, 265-290 7263 T
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888
G,-activated Calcium Transporter
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