Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641
Submitted 2 December 2003 ; accepted in final form 3 June 2004
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ABSTRACT |
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purinergic receptor; internalization; patch clamp
Another class of purinergic receptors is the P2X family of ATP-gated ion channels. Heterologous expression of six of the seven mammalian family members results in homomeric channels that are permeable to small monovalent and divalent cations, whereas one member (P2X6) contributes to functional cation channels mainly when coexpressed with other (P2X2 or P2X4) P2X subunits (38). In chicks, the P2X5 receptor appears to be permeable to both cations and Cl (38), and recent reports indicate that a P2X receptor may directly gate a Cl current in mouse parotid acinar cells (4). In a subset of the P2X family (homomeric P2X2, P2X4, and P2X7 and heteromeric P2X2,3), repeated or prolonged exposure to ATP leads to gradual opening of a pore that is highly permeable to large organic cations (up to 900 Da) such as N-methyl-D-glucamine (NMDG+) and YO-PRO-1 (25, 53, 54). In macrophages (20) and in cells transfected with P2X7 receptors, prolonged activation of the pore may lead to apoptosis or necrosis (58). The mRNA transcripts for three (P2X3, P2X4, and P2X5) of the P2X receptor subunits are present in FRTL-5 (17), and immunoreactivity for these same P2X subunits has been observed in rat thyroid follicular cells (18). At present, however, there are no reports of functional P2X receptor activity in thyrocyte-derived cells. In this study on FRTL cells, the parent cell line of FRTL-5, we show that ATP activates a plasma membrane cationic current and stimulates pronounced plasma membrane trafficking via activation of a P2X7 receptor that does not undergo pore formation.
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MATERIALS AND METHODS |
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Whole cell voltage clamp.
The cells were voltage clamped using an EPC7 patch-clamp amplifier (Adams List, Great Neck, NY) and the ruptured patch technique of whole cell recording (21). Data acquisition and analyses were performed using Pulse-HEKA software and the ITC-18 computer interface (Instrutech, Port Washington, NY). The bath was grounded with a Ag-AgCl pellet via a 1 M KCl agar bridge. Bath temperature was maintained by heating a 100-µl chamber to 36°C with a feedback regulator (Warner Instruments, Hamden, CT). A small coverslip chip containing cells was placed in the chamber, which was continuously perfused by gravity flow at a rate of 0.5 ml/min with solution prewarmed to 36°C. ATP, dissolved in bath solution, was applied to the cell by gravity flow from a large-bore pipette placed 340 µm from the cell. When reporting the effects of various antagonists on the ATP-evoked current, we divided current amplitudes by the cell capacitance to normalize for differences in cell size.
Solutions.
The pipette solution contained (in mM) 25 CsCl, 65 Cs2SO4, 2 EGTA, 0.6 mM CaCl2 (100 nM free Ca2+ at 36°C), 3 MgCl2, 10 NaCl, 10 glucose, 60 sucrose, and 10 HEPES (pH 7.4 at 36°C) and had an osmolarity of 305 mosmol/l. Cs+ was used as the major cation to block K+ currents in the cell. Standard bath solution consisted of (in mM) 150 NaCl, 5.6 KCl, 2 CaCl2, 2 MgCl2, 5 glucose, 30 sucrose, and 10 HEPES (pH 7.4 at 36°C) and had an osmolarity of 335 mosmol/l. Additions or modifications to the solutions were made by isosmotic ion substitutions and by using sucrose to make small adjustments in osmolarity. Concentrated stock solutions of 50 mM ATP or 5 mM UTP were prepared in Ca2+-free bath solution with reduced NaCl, subsequently adjusted for pH and osmolarity, and stored at 80°C. For experiments with no external Na+, the ATP stock solution was prepared with NMDG+ as the major cation. All experiments were performed at 3637°C. The concentrations of free Ca2+, Mg2+, and/or ATP4 were calculated with the software WEBMAXCLITE version 1.15 (available at http://www.stanford.edu/cpatton/maxc.html).
Confocal and epifluorescence imaging. Plasma membrane trafficking was assessed by continuously monitoring the fluorescence of FM1-43. The coverslip containing the cells was mounted in a chamber, rinsed with standard bath saline, and then maintained in 1 ml of bath saline containing 5 µM FM1-43 (added from a 5 mM stock solution prepared in DMSO). Agonists (ATP, UTP, or vehicle) were applied to the edge of the chamber either from a concentrated stock solution that was isosmotic in total solute concentration or by continuous perfusion of a small chamber with the indicated concentration of agonist. The solution temperature was maintained at 37°C by heating the chamber and the perfusion solution. To monitor plasma membrane permeability to YO-PRO-1, we continuously monitored fluorescence at 37°C in the presence of 2 or 5 µM YO-PRO-1.
To obtain measurements from only internalized membrane, the coverslip attached cells were prerinsed four times with standard saline at 37°C, incubated for 10 min at 37°C in saline containing 5 µM FM1-43 with or without agonists, and then washed four times at 37°C. The washes remove agonist, external dye, and dye bound to the plasma membrane. The cells were then examined for internalized fluorescence in standard saline at room temperature. For incubations performed at 4 and 23°C, the prerinse and wash were done at the same temperature as the incubation, but the fluorescence was measured at room temperature. When the effects of antagonists [except adenosine 5'-triphosphate-2',3'-dialdehyde (oxoATP)] were studied, the antagonists were present in the prerinse and during the incubation with FM1-43. The effects of oxoATP were studied by preincubating the cells with the drug for 2 h at 37°C, 5% CO2 in Coon's F-12 medium without serum or additives. The oxoATP was then removed and was not present during the incubation with agonist.
To measure the response to multiple applications of ATP, cells were rinsed, incubated for 10 min with or without 500 µM ATP, rinsed, allowed to recover for 10 min, incubated for 10 min in 5 µM FM1-43 in the presence or absence of ATP, rinsed free of ATP and FM1-43, and then monitored for internalized fluorescence. All manipulations were performed at 37°C except for the measurement of fluorescence, which was done at room temperature.
The confocal and epifluorescence measurements were performed on Nikon Eclipse 200 inverted microscopes (Nikon Instruments, Melville, NY) equipped with Photometrics CoolSnap HQ charge-coupled device (CCD) cameras (Roper Scientific, Tucson, AZ), Nikon sfluor x40 and x60 oil objectives (1.3 and 1.4 NA, respectively), and optical filters from Chroma Technology (Brattleboro, VT). Confocal images were obtained using the Noran 0Z video-rate laser scanner (Noran Instruments, Middleton, WI), argon-krypton laser, 488LP dichroic mirror, 500 LP emission filter, and Intervision acquisition software. Epifluorescence images were obtained using a xenon lamp, HQ475/30x excitation filter, Q495LP dichroic mirror, HQ515/30m emission filter, and MetaFluor or MetaMorph software (Universal Imaging, Downington, PA). The same filter combinations and excitation wavelengths were used to monitor FM1-43 and YO-PRO-1 fluorescence. Image analysis was performed off-line using MetaMorph software. The cell boundary was drawn from the transmission image, and then the mean pixel density of the inscribed area in the fluorescence image was determined. The pixel density per cell, after subtraction of the pixel density from regions devoid of cells, is reported as the relative fluorescence intensity per cell. Measurements were made in the linear range of the CCD camera by making adjustments in the acquisition time (25, 50, or 100 ms) and final scaling of the signal to 50 ms.
Cell surface biotinylation and Western blot. Jurkat cells in suspension culture and FRTL cells suspended by trituration were rinsed with cold phosphate-buffered saline containing 0.1 mM CaCl2 and 1 mM MgCl2, pH 9 (PBS-CM). Cell surface proteins were biotinylated at room temperature for 30 min with 1.5 mg/ml EZ-Link sulfo-NHS-LC-biotin (Pierce, Rockford, IL). The cells were rinsed, incubated at 4°C for 20 min with quench buffer (PBS-CM + 100 mM glycine, pH 9.0), rinsed, and then treated for 30 min at 4°C with lysis buffer (1% Triton X-100, 10 mM Tris, 150 mM NaCl, 5 mM EDTA, 10 µg/ml pepstatin, 10 µg/ml leupeptin, and 0.5 mM PMSF). The lysed cells were centrifuged, and the solubilized, biotinylated proteins in the supernatant were incubated overnight at 4°C with ImmunoPure immobilized streptavidin beads (Pierce). The beads were washed sequentially with lysis buffer, high-salt buffer (0.1% Triton X-100, 10 mM Trizma base, and 500 mM NaCl, pH 7.5), and no-salt buffer (50 mM Trizma base, pH 7.5) and were then heated to 85°C for 10 min in SDS sample buffer [50 mM Tris·HCl, pH 7.0, 1% (vol/vol) glycerol, 4% (wt/vol) SDS, 2% (vol/vol) 2-mercaptoethanol, and 0.13 mg/ml Coomassie blue R-250]. The beads were removed by centrifugation, and the eluted samples and MagicMark Western standards (Invitrogen, Carlsbad, CA) were separated by SDS-PAGE on Tricine gels containing 7.5% polyacrylamide (40). For Western blot analysis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked overnight at 4°C with PBS-CM containing 0.05% Tween 20 and 0.5% nonfat dry milk (PBS-TM) and were then incubated for 2 h at room temperature with rabbit anti-P2X7 polyclonal antibody (4 µg/ml) in PBS-TM. After being washed, the blot was exposed for 1 h at room temperature to horseradish peroxidase-linked donkey anti-rabbit Ig (Amersham, Piscataway, NJ) that was diluted 1:5,000 in PBS-TM. Antibody binding was detected by enhanced chemiluminescence with SuperSignal West Dura extended duration substrate (Pierce).
The primary antibody used for Western blot analysis was affinity-purified rabbit anti-P2X7 receptor that was raised against a synthetic peptide corresponding to extracellular residues 136152 of mouse P2X7 (Alomone Labs, Jerusalem, Israel). As a control, the blot was also probed with the same concentration of primary antibody that was preabsorbed by incubation for 1 h at 37°C with antigenic peptide (1 µg peptide/µg antibody).
Statistics. Results are reported as means ± SE; absence of error bars in the figures indicates an error that is too small for representation. Statistical significance (P < 0.05) was determined using Student's t-test when analyzing pairs or Kruskal-Wallis one-way ANOVA on ranks and Dunn's multiple comparison test (SigmaStat 2.0 software; SPSS, Chicago, IL).
Chemicals.
Hydrocortisone, bovine apotransferrin, bovine insulin, somatostatin, glycyl-L-histidyl-L-lysine acetate, calf serum from donor herd, ATP sodium salt, guanosine 5'-O-(2-thiodiphosphate) (GDPS) trilithium salt, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS) tetrasodium salt, ADP sodium salt,
,
-methyleneadenosine 5'-triphosphate (
-MeATP) lithium salt, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) trimethylammonium salt, brilliant blue G sodium salt (BBG), and oxoATP sodium salt were obtained from Sigma (St. Louis, MO). 2-Methylthioadenosine 5'-triphosphate (2-MeSATP) tetrasodium salt and UTP sodium salt were obtained from Calbiochem (La Jolla, CA), YO-PRO-1 diiodide and FM1-43 dibromide were obtained from Molecular Probes (Eugene, OR), and TSH was provided by Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA).
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RESULTS |
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Pharmacology of ATP-induced membrane internalization.
Pharmacological studies were performed under conditions that permit measurement of only the internalized fluorescence. P2X and/or P2Y receptor agonists such as 500 µM UTP, 2-MeSATP, ADP, and -MeATP were relatively ineffective compared with ATP (Fig. 7A). BzATP (500 µM), however, stimulated internalization to a greater extent than 500 µM ATP. Antagonists that inhibited the ATP-stimulated internalization were 100 µM PPADS, 50 µM Cu2+, 5 µM BBG, or a 2-h preincubation with 200 µM oxoATP (Fig. 7B). The effects of Zn2+ were also tested (Fig. 7C). At 20 µM, Zn2+ had little effect on ATP-stimulated internalization, but dose-dependent inhibition was observed at 100 and 500 µM. Zn2+ had no effect on basal activity (whether added at the time of FM1-43 incubation or included in the prewash). The effects of ATP on membrane internalization show the same pharmacological specificity as the activation of ionic current and are overall most consistent with activation of a P2X7 receptor.
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DISCUSSION |
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ATP activation of a P2X receptor-gated cation channel.
In whole cell, ruptured-patch recording of FRTL cells, prolonged (5 min) application of ATP activated a long-lasting, but rapidly reversible, cation-selective current. PPADS, an antagonist of most rat P2X (P2X1, P2X2, P2X3, P2X5, and P2X7) and P2Y (P2Y1, P2Y2, and P2Y6) receptors (44) inhibited the current. However, P2Y receptors were not involved, because UTP did not activate the ionic current and the ATP-activated current persisted when G protein activation was inhibited by inclusion of GDPS in the pipette solution. Current activation required high concentrations of ATP, >200 µM in our standard saline containing 2 mM Ca2+ and 2 mM Mg2+. Low affinity for ATP (EC50 = 0.11 mM) is a characteristic feature of the P2X7 receptor subtype, whereas a higher affinity (EC50 < 20 µM) is seen for P2Y and the other P2X subtypes (26, 46). BzATP was more effective than ATP in activating the current in FRTL. BzATP is an agonist of most P2X receptors (22, 39, 55) and of a few P2Y (P2Y2 and hP2Y11) receptors (13, 56). A relative potency of BzATP > ATP, however, is most characteristic of P2X7 receptors (38, 39). Both Cu2+ (50 µM) and Zn2+ (100 µM) inhibited the ATP-activated current in FRTL cells. Cu2+ inhibits ATP binding to P2X7 and P2X4 and potentiates current at P2X2 receptor subtypes (12, 53). Zn2+ (2100 µM) potentiates homomeric P2X2, P2X3, P2X4, and P2X5 and heteromeric P2X2/6 and P2X4/6 receptors, with an additional inhibitory effect occurring at higher concentrations of Zn2+ (10, 12, 29, 53, 55). In contrast, only the inhibitory effect is seen with P2X1 (55) and P2X7 receptors (53). Finally, preincubation with 200 µM oxoATP irreversibly inhibited the current. OxoATP is an irreversible inhibitor of P2X7 but a reversible inhibitor of P2X1 and P2X2 receptors (39). Overall, the long-lasting nature of the current and the pharmacological profile are most consistent with the involvement of a P2X7 receptor. The P2X7 subtype is unique in its inability to form heteromers with other P2X subunits (38).
Pore activation is a common (25, 38, 53), but not obligatory (20, 43, 45), property of heterologously expressed and endogenous P2X7 receptors. In FRTL cells, the ATP-activated receptor remained relatively impermeant to NMDG+ and YO-PRO-1 during a 5- to 10-min exposure to ATP at 37°C (in the presence or absence of divalent cations). Thus, as in bovine endothelial cells (45), human retina glial cells (41), and parotid acinar and duct cells (33), ATP activates a P2X7-like receptor channel in FRTL without large pore formation. This could be due to absence of an accessory protein (47) or a variation of the COOH terminus of the P2X7 receptor that is essential for pore activation (51).
The ATP-activated current increases in amplitude and/or grows with successive applications of 500 µM ATP or increases upon the first application of 2 mM ATP. The growth is not due to slow activation of pores because the channel remains impermeant to NMDG+. Furthermore, ion selectivity does not change during the rising phase of the current (Kochukov MY and Ritchie AK, unpublished observations). Such P2X7 receptor kinetics have been observed by others and have been proposed to be due to a slow increase in potency with submaximal concentrations of ATP (23) and/or a slow assembly step involving the cytoskeleton that is required for channel gating (33).
A P2X7-like receptor mediates ATP stimulation of plasma membrane trafficking. Confocal and epifluorescence measurements of the cell surface marker FM1-43 showed that ATP stimulated a very large increase in cell-associated fluorescence with dramatic labeling of internal membranes. Although this labeling pattern could occur secondary to permeation of FM1-43 through an ATP-activated pore, this seems unlikely because ATP did not increase permeability to YO-PRO-1. FM1-43 influx may also enter cells via nonselective cation channels, as occurs in sensory hair cells (36). FM1-43 influx through such channels, however, is characterized by rapid kinetics (in seconds) and influx at 4°C. In contrast, the ATP-induced fluorescence changes in FRTL began after a delay of several minutes, gradually increased over the next 11 min, was markedly attenuated at 23°C, and was completely absent at 4°C. The slow kinetics and temperature sensitivity are consistent with ATP stimulation of membrane trafficking in which the increase in fluorescence is due to exocytotic fusion of internal membranes with the plasma membrane and the pronounced labeling of internal membranes is due to increased internalization of dye-labeled plasma membrane.
BzATP was a more potent stimulator of membrane internalization than ATP, whereas agonists such as 2-MeSATP (9, 57), -MeATP, and ADP (39), which are ineffective or less effective than ATP (39) at P2X7 receptors, had relatively little effect on membrane internalization. The response to ATP was inhibited by PPADS, oxoATP, Zn2+, Cu2+, and 5 µM BBG (38). At 5 µM, BBG inhibits P2X7 and P2X2, but much higher concentrations are needed to inhibit P2X1, P2X2, P2X3, P2X2/3, P2X4, and P2X1/5 receptors. Two distinguishing features of P2X7 receptors, low affinity for ATP and potentiation upon removal of extracellular Ca2+ and Mg2+, were also present. The EC50 value for ATP stimulation of FM1-43 internalization was
440 µM in standard saline and 14 times lower (EC50
33 µM) in nominally Ca2+-free, low-Mg2+ saline. This is consistent with other reports that the active ligand for the P2X7 receptor is the fully ionic ATP4 rather than ATP that is complexed with a divalent cation (30, 38). Finally, replacement of Na+ with NMDG+, which enhances the potency of ATP (37) for P2X7 receptors, also greatly enhanced the effect of ATP on membrane internalization in FRTL. The pharmacological profile indicates that ATP stimulation of membrane internalization is mediated by a P2X7 receptor. Although P2X7 mRNA transcripts were not detected in the FRTL-5 subclone (17), in FRTL we detected P2X7 receptor immunoreactivity using Western blot analysis.
Transduction mechanism.
The mechanism by which P2X receptor activation results in stimulation of exocytosis and endocytosis in FRTL is unknown. Because the stimulation of trafficking persists in Ca2+-free (with 2 mM EGTA), low-Mg2+ (20 µM), or Na+-free solutions, these actions of ATP occur independently of the influx of ions through the receptor-gated channel. A Ca2+-independent effect of extracellular ATP on plasma membrane trafficking has been previously observed in rat brown adipocytes (31); however, the receptor in adipocytes does not appear to involve P2X7 receptors, because it is activated by low concentrations of ATP, ADP, and 2-MeSATP (42). Some previously reported Ca2+-independent actions of P2X7 receptors in other cell types include stimulation of MAP kinases (3) and the Ca2+-insensitive forms of PLA2 (1) and PLD (24). MAP kinases (27), PLA2 (6), and PLD (11) are known to affect plasma membrane trafficking. The P2X7 receptor forms a complex with a number of membrane, extracellular matrix, and intracellular proteins that could be involved in signaling from the receptor to the cytoskeleton or other scaffolding proteins involved in trafficking (28, 58).
Function.
The P2X7 subtype is highly expressed in many epithelial and hematopoietic cells, where it activates a number of signaling cascades. It appears to be a regulator of inflammation, because some of the activities evoked by the receptor include T-cell activation and maturation and killing of invading microorganisms via apoptotic death of macrophages (38). In monocytes, activation of P2X7 receptors mediates rapid secretion of IL-1 via plasma membrane microvesicle shedding, a process opposite to the pronounced stimulation of plasma membrane internalization that we observed in FRTL cells (34). The function of the P2X7 receptor in stimulation of membrane trafficking in FRTL is unknown. Because the stimulation requires very high concentrations of ATP, it is unlikely to play a physiological role in the exocytosis and internalization of thyroglobulin. We speculate that activation by high concentrations of ATP may reflect a response to ATP released from damaged cells that could influence the uptake of nutrients, or changes in the composition of membrane proteins such as channels or transporters, of nearby healthy cells.
In summary, ATP activates a nonselective cation conductance and plasma membrane trafficking via a P2X7 receptor that does not undergo pore expansion. The stimulation of internalization does not involve Na+ or Ca2+ influx; hence, P2X receptor activation may couple to other proteins that initiate the membrane trafficking independently of channel activity. This is the first evidence of functional P2X receptors in cells of thyrocyte origin.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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