Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901
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ABSTRACT |
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ATP activates an apical-to-basolateral
nonselective cation current across the skin of larval bullfrogs
(Rana catesbeiana) with similarities to currents carried by
some P2X receptors. A functional P2X receptor was cloned from tadpole
skin RNA that encodes a 409-amino acid protein with highest protein
homology to cP2X8. RT-PCR showed that this transcript was
found in skin, heart, eye, brain, and skeletal muscle of tadpoles but
not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP
activated a transient current similar to other P2X receptors and the
ATP-activated transient in short-circuit current
(Isc) across intact skin. The agonists
2-methylthio-ATP and adenosine-5'-O-(thiotriphoshate) also
activated transient currents. ,
-Methylene-ATP and ADP were poor
agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor
to agonist activation. Under external Na+-free,
Ca2+/Mg2+-free conditions
(N-methyl-D-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated Isc in the
tadpole skin. Because the clone is not found in adult skin, it may have
some exclusive role in the tadpole such as sensory reception by the
skin or triggering apoptosis at metamorphosis.
tadpole; P2X receptor clone; purinergic; amiloride; epithelium
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INTRODUCTION |
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ION TRANSPORT STUDIES on the skin of the larval bullfrog (Rana catesbeiana) show that ATP activates a nonselective apical-to-basolateral cation short-circuit current (Isc). This current, under continuous ATP application, exhibits rapid activation and subsequent desensitization. Furthermore, suramin, a purinergic blocker, inhibits the ATP-activated current in tadpole skin (9). Together, these properties suggest participation of channels related to the P2X receptor family (4).
The P2X receptor family represents a group of nonselective cation channels that are activated by ATP. Currently, there are at least seven subtypes in this group (4, 14, 27). Recently, an eighth subtype was reported (1). Various combinations of these receptors form homomeric channels as well as heteromeric hybrid channels (27, 32). They are distributed in a number of tissues, including neurons, smooth muscle, several types of epithelia, and macrophages. The exact physiological role of these receptors is still unclear. Recent evidence suggests that P2X1 receptors in humans may have a function in platelet aggregation, although the mechanism behind this is still not well defined (28). Similarly, a gene knockout study of P2X1 in rats showed that this receptor has an important function in reproduction in males (26). P2XM, a related clone expressed primarily in skeletal muscle, may play a role in differentiation or proliferation of skeletal muscle (34). In a variety of epithelia, activation of P2X receptors leads to activation of a chloride-dependent Isc (24, 25, 30), activation or inhibition of a sodium-dependent Isc (9, 25), and activation of cilia in airway epithelia (22). Thus the P2X family apparently has a number of diverse physiological roles.
Ligand-activated transport across the larval bullfrog skin has been studied by using the short-circuit current technique including fluctuation analysis (9, 11, 12, 13, 18). There is currently no information as to what proteins are involved, but as mentioned above, P2X receptors are likely candidates (9). In this study, we show that P2X receptors cloned from larval skin expressed in Xenopus oocytes respond to ATP with many characteristics in common with larval bullfrog skin. Our data indicate that this receptor has the greatest homology with and similarity to the chick embryo P2X8 clone (cP2X8) (1).
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METHODS |
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RNA extraction. Tadpoles were anesthetized, and whole skins were removed from the tadpole body. The basolateral sides of the skins were scraped with a scalpel to remove any attached tissue. Skin cells were then removed by washing in an EDTA-Ca2+/Mg2+-free solution (71.9 mM NaCl, 3.8 mM KCl, 3.9 mM Na2HPO4, 1.1 mM glucose, 10 mM HEPES, and 2.5 mM EDTA, pH 7.4) for 5-10 min (10). Dissociated cells were separated using a stainless steel mesh. The skin cells were pelleted by centrifugation and homogenized in Trizol reagent (Life Technologies). RNA was extracted from the skin cells with Trizol reagent by following the manufacturer's protocol.
P2X fragment isolation from skin RNA. Total RNA (5 µg) was reverse transcribed with SuperScript II (Life Technologies) to cDNA by using random primers and following the manufacturer's suggested reaction conditions in a 20-µl reaction. Four degenerate primers (Operon) were then used in a nested reverse transcription-polymerase chain reaction (RT-PCR) approach with Taq polymerase (Perkin Elmer). One-tenth of each RT reaction was used in the first round of PCR with the sense degenerate primer 5'-TGYGARRTNTYNGTRCANTCNCCRTA-3' (5) and the antisense degenerate primer 5'-AAYTTNCCNGCNTKNCCRTCNAC-3' in a 50-µl reaction. The first-round product (2 µl) was used in a second round of PCR with the sense degenerate primer 5'-GCNGARAAYTTYACNYTNTTYATHAARAA-3' and the anti- sense degenerate primer 5'-GCRAAYCTRAARTTRWANCC-3' (5). The first round of PCR ran 40 cycles at a 55°C annealing temperature for 45 s, a 72°C extension temperature for 90 s, and a 94°C denaturation temperature for 60 s. The second round of PCR ran 35 cycles at similar conditions.
Cloning of the P2X receptor.
The PCR fragment obtained from degenerate primers was randomly labeled
with [-32P]ATP and used to screen a Lambda ZAP
(Stratagene) cDNA library made from tadpole tail RNA (generously
provided by Valerie Anne Galton and Mark Schneider, Dartmouth College,
Hanover, NH). Four positive plaques were isolated, pBluescript SK(
)
phagemid was isolated with ExAssist helper phage (Stratagene), and the
phagemid was transformed into Escherichia coli for
amplification. Inserts were sequenced in both directions. Sequencing
showed that the cDNA clone was not full length at the 5' end. Three
reverse primers (reverse primer 1,
5'-GTAGTCCGTCTCATTCACATC-3'; reverse primer 2,
5TTCCAATAACACCACCCTCC-3'; and reverse primer 3,
5'-GAAGATGGGGCATAATG-3') were designed to the library fragment and used
in 5' RACE (rapid amplification of cDNA ends) (16) to
obtain the remainder of the 5' sequence. Reverse primer 1 was used for the RT reaction with the Thermoscript kit (Life
Technologies) according to the manufacturer's suggested reaction
conditions. The RT product was then treated with RNase A and RNase H
and purified over a column (Qiagen) to remove degraded RNA and leftover
primers. A 5' poly(A) tail was added to the 5' end with Terminal
Transferase (Life Technologies), and 2 µl of the tailed RT was used
in a second-strand synthesis and first-round PCR reaction.
Second-strand synthesis was primed by a poly(dT) primer that also
had an anchor sequence attached (5'-AACGGTTCCTTGAACCTGTCGACTTTCCTT- TTTTTTTTTTTTTTTTTT-3'), and the
first-round PCR reaction used reverse primer 2 and anchor primer 1 (5'-AACGGTTCCTTGAACCTGTC-3'). The second-strand synthesis ran
at a 48°C annealing temperature for 120 s, followed by a 72°C extension for 50 min. The first round of PCR ran 35 cycles at a 55°C
annealing temperature for 60 s, a 72°C extension temperature for
120 s, and a 94°C denaturation temperature for 45 s.
Second-strand synthesis and the first round of PCR were carried out in
the same tube. The 50-µl first-round reaction (2 µl) was used in a
second-round nested PCR reaction that used reverse primer 3 and a second anchor primer (5'-CCTTGAACCTGTCGACTTTCC-3'). The
second-round reaction ran 35 cycles at a 55°C annealing temperature
for 60 s, a 72°C extension temperature for 120 s, and a
94°C denaturation temperature for 45 s. Both first and second
rounds used Taq polymerase and proceeded according to the
suggested manufacturer's reaction conditions. Two independent 5' RACE
products were sequenced from which a start primer
(5'-AATGTGAGCAGATAGAATGGGG-3') and an antisense primer (5'-TGTCCAGGTTACAATCCCATTC-3') were designed. These primers were used
in three independent PCR reactions that ran 30 cycles at a 58°C
annealing temperature for 30 s, a 72°C extension temperature for
60 s, and a 94°C denaturation temperature for 30 s
with Pfu polymerase (Stratagene) according to the suggested
manufacturer's reaction conditions. One cDNA fragment from each of the
three PCR reactions was sequenced to determine a correct consensus
sequence and eliminate introduction of PCR errors when the full-length clone was assembled. The cDNA library fragment and the fragment generated with Pfu polymerase were then digested with
SalI and ligated together. The final cDNA was sequenced to
verify that no mutations were introduced relative to the consensus sequence.
Tissue distribution.
Tadpoles were anesthetized, and total RNA was collected from several
tissues of the tadpole with Trizol reagent. Total RNA (4.5 µg) from
each tissue was reverse transcribed by using SuperScript II primed with
poly(dT) (12- to 18-mer). Ten percent of each RT reaction was used in a
PCR reaction. Two different PCR reactions were made for each tissue:
one used gene-specific primers for the P2X clone (sense
5'-ATGATTATTGCCCCATCTTCCAC-3' and antisense 5'-TGCTTTACGCCTCCACACTC-3'); the other used gene-specific primers for
-actin (sense 5'-CCTGAAGAACACCCTGTCCTTCTC-3' and antisense 5'-TGTCACGCACGATTTCTCTTTCTG-3') to show approximate levels and to
indicate that RT occurred. Both reactions were run 35 cycles at a
65°C annealing temperature for 30 s, a 72°C extension
temperature for 60 s, and a 94°C denaturation temperature for
30 s. Aliquots of each reaction (20 µl) were run down a 1.5%
agarose gel and visualized with ethidium bromide staining.
Whole cell clamp.
Capped cRNA was synthesized from linearized plasmid DNA with a T3
MessageMachine kit (Ambion). Xenopus oocytes were surgically removed and defolliculated in 2 mg/ml collagenase (Sigma) in OR-2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM
HEPES, pH 7.5). cRNA (50 nl of 0.3 µg/µl) or H2O (50 nl) was injected into each defolliculated oocyte by using a Drummond
injector. Oocytes were incubated for 24 h at 18°C in ND-96 (96 mM
NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) supplemented with pyruvate, penicillin, and
streptomycin. Voltage-clamp experiments were done with a two-electrode
voltage clamp (model 8500; Dagan, Minneapolis, MN), and the data were
recorded by a computer. Oocytes were analyzed under varying conditions
but were always clamped at
80 mV. For Ca2+- or
Mg2+-free experiments, Ca2+ and/or
Mg2+ were replaced by 0.5 mM EDTA. For Na+
replacements, N-methyl-D-glucamine (NMDG) and/or
K+ was exchanged for Na+ at the same molar
concentration. Data were analyzed with Origin 6.0 (Microcal) software.
Fluorescence with propidium iodide. Injected oocytes (P2X or H2O) were placed in ND-96 solution (1.8 mM Ca2+ or Ca2+ free) that contained 10 µM propidium iodide. ATP (10 µM) was added, and fluorescence was determined 30 s after ATP application. Fluorescence was then photographed every minute for 6 min with an Olympus inverted microscope and a digital camera interfaced with a computer.
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RESULTS |
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Cloning of the tadpole skin P2X receptor.
A 344-bp cDNA fragment was isolated by using a nested degenerate RT-PCR
approach (see METHODS). This fragment had the closest translated amino acid sequence homology to human P2X5
(hP2X5). This fragment was radiolabeled and used to
screen a tadpole tail cDNA library by hybridization. From that screen,
a 1,600-bp clone was isolated that, after sequencing, was shown to be
only one-half of the coding region for P2X receptors. 5' RACE was
employed to obtain the final full-length clone (fP2X8) of
2,423 bp (Fig. 1). The predicted
translated amino acid sequence of the clone shows a 409-amino acid
protein, which has 10 conserved cysteines and 2 putative transmembrane
regions consistent with other members of the P2X family
(1). Figure 2 represents a
comparison of the protein structure of fP2X8,
cP2X8, and rat P2X5 (rP2X5). The amino acid sequence of the tadpole clone has ~74% homology with cP2X8 and ~67% homology with rP2X5 (Fig. 2).
Homology between the sequences decreased after position 378.
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Tissue distribution.
Analysis of tadpole total RNA by RT-PCR showed that P2X expression was
not unique to skin RNA (Fig. 3), because
PCR products were found in brain, heart, muscle, and eye tissue. No
products were found in the liver or intestine. -Actin products were
found in all tissues demonstrating successful RT of all samples.
Interestingly, no product was produced from the RNA of adult skin (Fig.
3) or from brain or heart (data not shown).
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Electrophysiological properties of the fP2X8 clone.
After injection of in vitro cRNA into Xenopus oocytes,
classic ATP-activated currents were observed under whole cell clamp conditions. ATP (40 µM, continuous application) stimulated a fast inward current that rapidly desensitized (Fig.
4A). ATP application to
H2O-injected oocytes showed no activation on the current
scale used to record from P2X-injected oocytes (Fig. 4B).
However, repeated application of ATP showed a pronounced degradation of
the response, often referred to as rundown. Figure 4C shows
the results of successive ATP applications with 5 min of washout in
between. The ATP response decreased by ~40-90% with each
application until it could no longer be detected. Increased time
between exposure to ATP decreased the rundown; however, recovery was
variable such that experiments that would require multiple ATP
applications on the same injected oocyte could not accurately be
performed. Twenty minutes between ATP application gave a rundown that
varied from 30-65% of the previous response (Fig. 4D).
Lower concentrations of ATP (1 µM) yielded smaller currents.
Nonetheless, successive applications of ATP at that concentration also
produced similar rundown. Rundown has been found in some of the other
P2X receptor channels, including P2X1 and cP2X8
(1, 33). Of these P2X subtypes, it appears the
cP2X8 rundown is the most dramatic (1).
Because of this rundown, accurate dose-response curve values for ATP
and other ligands could not be determined.
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Fluorescent dye uptake through fP2X8.
Some of the P2X receptors, including P2X2,
P2X4, and P2X7, have the ability to form a
large pore size under low divalent (0.1 mM) conditions (20,
35). This has been clearly shown by the ability of large
molecules like YO-PRO1 to enter cells expressing these channels. Under
low divalent conditions, the fP2X8 receptor had
ATP-activated currents in NMDG, Na+-free solutions.
Therefore, the ability of a large ion like propidium to enter the
injected oocyte was also studied. An fP2X8-injected oocyte
showed an obvious increase in fluorescence after 10 µM ATP
application in Ca2+-free solution (Fig.
8). Time 0 represents 30 s after ATP application, and fluorescence was measured at 1-min
intervals thereafter. Similar results were obtained using nine other
oocytes. However, when the same experiment was run in 1.8 mM
Ca2+, no increase in fluorescence was observed
(n = 8). H2O-injected oocytes showed no
increase in fluorescence when treated with ATP in either divalent-free
or 1.8 mM Ca2+ solutions. The fluorescent spot on the sham
oocytes was the site of injection and was observed on all injected
oocytes.
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DISCUSSION |
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ATP application to the skin of the larval bullfrog induces a transient inward (apical to basolateral) nonselective cation current. The time course and pharmacology of this current are similar to some P2X receptors (9). Therefore, we decided to look for molecular evidence that P2X receptor transcripts existed in the tadpole skin RNA. We isolated and characterized a functional P2X receptor from the larval bullfrog skin that encodes a 409-amino acid-long protein that contains 10 conserved cysteines and 2 putative transmembrane regions. It is structurally most closely related to cP2X8 and rP2X5. cRNA was synthesized from this clone and injected into Xenopus oocytes. Under whole cell clamp conditions, injected oocytes showed classic P2X responses to ATP. We have tentatively named this clone fP2X8.
The time courses after ATP activation and responses to agonists and
blockers have been used to classify P2X receptors into various
subgroups. This tadpole channel is blocked by both PPADS and suramin,
which is characteristic of P2X1, P2X3,
P2X5, and cP2X8 (1, 27). Long-term
blockage by PPADS is a characteristic of P2X5
(17). The tadpole channel is activated by ATPS and 2-MeS-ATP similarly to cP2X8 (1) and other P2X
receptors (27). Under low-Ca2+ conditions,
fP2X8 undergoes a biphasic response similar to that observed with P2X4 and cP2X8. Additionally,
both the tadpole clone and cP2X8 have a marked rundown that
appears to be the most severe of any of the P2X receptors. It appears
that this channel has a number of similarities to cP2X8.
One exception is that fP2X8 receptor is insensitive to
,
-MeATP, one of the major agonists used to classify P2X
receptors. A second difference between cP2X8 and
fP2X8 receptor is the relatively irreversible block caused by PPADS of the latter. PPADS does block the ATP response of
cP2X8, but it appears to readily reverse. P2X5
has a partially irreversible blockade with PPADS (17). In
that case, after PPADS washout there was an attenuated ATP response.
Interestingly, the lysine at position 251, which is hypothesized to be
involved in the irreversible PPADS blockage of P2X5
(6), is a glutamic acid residue in fP2X8 and
cP2X8. The next closest lysine for fP2X8 is at
position 236, which is an asparagine and a serine for cP2X8
and rP2X5, respectively. This finding suggests that
lysine-236 may be an important structural position for PPADS binding.
At the protein level, this P2X receptor has closest homology to cP2X8 (74%) followed by rP2X5 (67%). The homology increases to 80% for cP2X8 and 74% for rP2X5 if the amino acids after position 378 are not utilized in the comparison. Because both fP2X8 and cP2X8 are homologous to P2X5 until position 378, it may be possible that fP2X8 and cP2X8 are actually splice variants of P2X5. For example, when the COOH terminus of rP2X5 was spliced onto the missing COOH terminus of hP2X5, this channel appeared to exhibit some of the properties of cP2X8, including the transient current and the marked rundown (23). Some natural COOH-terminal splice variants have been found for P2X2 (2). Currently, no naturally occurring splice variants for P2X5 have been reported that provide characteristics different from those of the functional P2X5 clone previously described.
The tissue distributions of cP2X8 and fP2X8 are also quite similar. The transcripts for both clones were found mainly in excitable tissues. It is also interesting that both fP2X8 and cP2X8 were cloned from developmental stages of the organism in question.
The original purpose of this experiment was to identify a P2X receptor involved in Isc activation of the tadpole skin. The receptor we have cloned shares the following characteristics with ATP-activated cation transport across tadpole skin. The time course and phenotype of the response of both the tadpole skin and the cloned receptor show transient currents that rapidly desensitize even with ATP still present. Suramin blocks both the skin response and the cloned receptor. We also found (19) that aldosterone treatment increases the ATP-activated tadpole response and increases the amount of fP2X8 transcripts found in treated compared with untreated skin cells. It is apparent that the fP2X8 channel cannot account for the entire tadpole response since it was not activated by amiloride and ACh and is not blocked by W-7 (9, 11-13). Furthermore, the tadpole Isc response does not have the dramatic rundown that the cloned receptor does. There may be other P2X receptors in the skin that form heterodimeric channels that may eliminate the degree of rundown found, as in the case of the P2X5-P2X1 heterodimer (33). This also may be the case in the tadpole skin. Moreover, other proteins may also be involved in the response such as nicotinic channels, which have been recently shown to interact with P2X channels (21).
Physiological and molecular evidence for P2X receptors has been found in human and mouse pulmonary epithelia (30), rabbit airway epithelia (22), pancreatic duct cells (24), parotid acinar cells (31), a mouse renal cell line (25), and LLC-PK1 cells (15). To our knowledge, the present study represents the first complete cloning and characterization of a P2X receptor from a Na+-transporting epithelium. At present, we can only speculate about possible physiological roles for this channel. Because P2X3 and others may participate in pain reception, a sensory role for fP2X8 is a possibility in the tadpole. This may include detecting predators, food, or other substances in the water. Another possibility may be that this channel is used to sense tissue damage. P2X3 is believed to have a role in nociception through channel activation by ATP released from damaged cells (7). Furthermore, some P2X receptors have been linked to apoptotic pathways in dendritic cells. This may occur through the large pore formation with ATP activation (8). The fP2X8 channel also forms a large pore under low divalent conditions that would most likely lead to cell death. Because there is extensive apoptosis during tail reabsorption at metamorphosis, fP2X8 could be involved. Even though Brodin and Nielsen (3) have identified a probable P2X receptor in adult frog skin, we were unable to find fP2X8 transcripts in adult skin, heart, or brain using RT-PCR. Apparently their ATP-gated channel is not related to fP2X8. The fact that fP2X8 was not detectable in adult frogs suggests that this channel may have specific roles in the larval amphibian.
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ACKNOWLEDGEMENTS |
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We thank Mark Schneider and Valerie Galton for providing samples of their cDNA library from tadpole tail.
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FOOTNOTES |
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Support for this work was provided by the American Heart Association, Midwest Affiliate.
Address for reprint requests and other correspondence: T. Cox, Dept. of Physiology, Southern Illinois Univ. School of Medicine, Carbondale, IL 62901 (E-mail: tcox{at}siumed.edu).
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.
Received 19 December 2000; accepted in final form 21 April 2001.
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