Ionotrophic ATP (P2X) Receptors
Alon Meir, Ph.D.
P2X receptors are surface membrane ligand-gated channel proteins that rapidly translate
extracellular ATP elevation into an intracellular Ca2+ surge which affects the cell’s
secretion, gene expression, contraction and migration capabilities as well as viability
and differentiation status. Here, we discuss recently published uses of Alomone Labs'
specific P2X antibodies in the growing field of ATP-gated channels.
Adenosine triphosphate (ATP) is an energy
storing molecule in all cells. However, in several
cellular systems, where it might be present
in the extracellular space, ATP also serves as
a neurotransmitter or hormone. Well studied
examples of such systems include inflammation
states, where the dead cell’s cytoplasmic content
or “inflammatory soup” that contains a high
level of ATP, affects lymphocytes and healthy
cells. Another example is the extracellular space
around synapses in the nervous system or around
innervated tissue; where synaptic vesicles release
neurotransmitters along with ATP (which exists
at high concentrations) upon their fusion with the
plasma membrane. In such cellular systems the
sensing of extracellular ATP, which is translated
by membrane receptors into a cytosolic signal,
enables the cell to react to “the inside is coming
from outside” signal.
The receptors for extracellular ATP are divided
into metabotrophic (P2Y) and ionotrophic (P2X)
receptors. P2Y receptors are G-Protein Coupled
Receptors (GPCRs, of which 8 isoforms exist) and
their downstream signaling is defined by the G
proteins they are complexed with. They usually
respond in a longer time range than P2X receptors
and are not discussed further here.
P2X receptors are protein complexes, composed
of three similar subunits that bear an ion channel
within a membrane spanning domain, which
gates open upon ATP binding (to an extracellular
domain). All seven P2X channel isoforms (P2X17) are permeable to organic mono and divalent
cations including Ca2+ (for review on purinergic
transmission, see reference 1).
The pharmacology of P2X receptor channels is
not very specific in differentiating between the
various isoforms. To date, specific antibodies are
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the most common tool used for indicating and
identifying the P2X subtype.
Figure 1. Expression of P2X4 Receptor
Alomone Labs specific P2X antibodies,
besides having an important role in showing
the localization of P2X channels, also assist in
deciphering their physiological and pathological
roles. We cite papers where these antibodies
were used: Immunoprecipitation (IP) experiments
to demonstrate physical interactions between
P2X isoforms and with other proteins2-7; Western
blot to demonstrate expression in cells or tissue
extracts, or knockdown by siRNA or in knockout
mice 3,5,6,8-18; Immunohistochemistry (IH) and
Immunocytochemistry (IC) experiments to localize
the expression sites and patterns of the channels
using different microscopic methods5,8,11-13,15,17,19-24.
Knock-out P2X4–/– Mice.
Channel in Wild-type P2X4+/+ but not in
Many studies explore the roles of P2X channels
in white blood cells and in tissues affected by
injury as well as in neurons and glia both in the
context of stress and injury and in response to
synaptically released ATP.
Using Anti-P2X1 (#APR-001), Anti-P2X2 (#APR003), Anti-P2X4 (#APR-002) and Anti-P2X7 (#APR004) antibodies, P2X1, 2, 4 and 7 were detected
in mouse urinary bladder, exemplifying the role
and expression of these subunits in smooth
muscle19. P2X2 was detected in pulmonary
neuroepithelial bodies (probably forming a
complex with P2X3)22.
A, C, E, Using Anti-P2X4 antibody (#APR-002), P2X4
subunit immunoreacts throughout the cell body layers of
hippocampus and is absent in knock-out mice (B, D, F). Pyr,
Pyramidal cell. G, H, Detection of β-galactosidase. 5-Bromo4-chloro-3-indolyl- β D-galactopyranoside (X-gal) staining in
hippocampus of knock-out P2X4–/– mice but not in wild-type
P2X4+/+ mice. Scale bars: A, B, G, H, 500 µm; C, D, 50 µm;
E, F, 40 µm. I, P2X4 subunit is immunoreactive in cerebellar
Purkinje cells and is absent in the knock-out P2X4–/– mice (J).
K, X-gal staining shows the expression of β-galactosidase
P2X3 channel was found to be expressed in
sensory neurons using Anti-P2X3 (#APR-016)
antibody5. Upon a decrease in Nerve Growth
Factor (NGF) levels, these neurons are induced
to express more P2X2/3 complexes, changing
ATP derived signaling. In such sensory neurons,
the expression of P2X3 was shown to depend
on calcitonin gene related peptide25. Using Anti
in cerebellar Purkinje cells (P), as well as some cells of the
granular layer (gl) and mossy layer (ml) from knock-out
P2X4–/– mice. L, P2X4 subunit immunoreactivity in acinar and
ductal cells of the submandibular gland is completely absent
in knock-out P2X4–/– mice (M). N, Extensive expression of
β-galactosidase in submandibular gland of knock-out
P2X4–/– mice. Scale bars: I, J, K, 20 µm; L, M, N, 50 µm.
Adapted from reference 17 with permission of The Society for
Neuroscience.
Modulator No.23 Fall 2009 www.alomone.com
Figure 2. Marked Upregulation of P2X4
elevation around the cell.
Levels in the Spinal Dorsal Horn Following
Using Anti-P2X7 antibodies (#APR-004 and/
or #APR-008, directed against intracellular and
extracellular epitopes, respectively), showed that
P2X7 channels are expressed in macrophages
where they control IL-β release9-11. Anti-P2X7
antibody was used to immunoprecipitate the
channel from macrophages, later to be blotted
with anti-Pannexin-1 antibody, suggesting a
Injury to the L5 Nerve.
P2X4
Nerve injury
Contralateral
Although P2X7 is absent in macrophages of
knockout mice, it was recently demonstrated,
that functional channels are expressed in T
lymphocytes of these mice (using #APR-004)11.
This paper probably resolves a controversy
regarding P2X7 antibody specific detection in
knockout mice. In addition to the expression of
Ipsilateral
Sham
Contralateral
physical and functional complex between the ATP
receptor and the gap junction channel3.
CFA
Ipsilateral
Contralateral
Ipsilateral
Visualization of the P2X4 protein detected by using AntiP2X4 antibody (#APR-002) in the L5 dorsal spinal cord by
Figure 3. in vivo Up-Regulation of P2X2,4 Proteins in Gerbil Hippocampus Following Ischemia.
immunofluorescence analysis with confocal microscopy.
Photographs show the P2X4 immunofluorescence in the
dorsal horn 14 days after nerve injury (top), 14 days after
sham operation (bottom left) and 7 days after the injection of
CFA into the plantar surface of the hindpaw, an inflammatory
pain model (bottom right). Scale bars, 200μm.
Adapted from reference 24 with permission of Nature
Publishing Group.
P2X4 antibody, it was demonstrated that P2X4
is expressed in many tissues7; the protein was
detected in brain, lung and submandibular gland
of wild type and absent in tissues from P2X4
knockout mice17 (Figure 1). In the CNS P2X4
was mainly in microglia16,18,24. It was shown to
be upregulated in spinal cord microglial cells
following the induction of nerve injury24 (Figure
2), and in hippocampal cells following ischemia16
(Figure 3). Morphine treatment enhances
microglial migration by increasing the expression
of P2X418. Expression of P2X4 and other P2X
receptor channels were detected in neurons
innervating the carotid body O2 chemoreceptors23
(Figure 4), suggesting a role in negative feedback
loop that inhibits ATP releasing chemoreceptors
(during hypoxic stress). P2X4 and other P2X
channels were also shown to be expressed in
chicken mesenchymal cell cultures15, suggesting a
role in bone development and repair.
P2X4 was shown to heteromerize with P2X7
subunits by co-immunoprecipitation in an
expression system 6. It was also shown for both
P2X4 and P2X2 that mutating the ATP binding
site causes reduced surface expression of the
mutated subunit14.
Nissl staining of the hippocampus of a sham-operated (A) or ischemic animal (B); P2X2 immunostaining (detected with AntiP2X2 antibody (#APR-003)) of a sham-operated (C) or ischemic animal (D); P2X4 immunostaining (detected with Anti-P2X4
P2X7 receptor channel differs from all the other
isoforms in that it is activated only by very high
ATP concentration and in many cases P2X7
channels mediate a very robust intracellular Ca2+
elevation1. Therefore, this receptor channel is
frequently implicated as a transducer of acute ATP
Modulator No.23 Fall 2009 www.alomone.com
antibody (#APR-002)) of a sham-operated (E) or ischemic animal (F); arrows: CA1–CA2 transition zone. (G) Higher magnification
of P2X2-immunolabeling of the CA1 region of an ischemic animal; arrows: network of fibers in the pyramidal cell layer and apical
dendrites. (H) Higher magnification of P2X4-immunolabeling of the CA1 pyramidal cell layer of an ischemic animal; arrows:
processes surrounding an unstained cellular body (marked by asterisk). (I) Higher magnification of P2X2-immunolabeling of the
CA1 pyramidal cell layer of an ischemic animal. Meshwork of fibers and puncta surrounding unstained cellular bodies marked by
asterisks. Scale BARS=100 μm (A–F); (G)=40 μm; (H, I)=10 μm.
Adapted from reference 16 with permission of Elsevier.
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P2X7 in glial satellite cells12, its expression was
demonstrated in neuronal nerve terminals13,20,21.
It was suggested that activation of P2X7 channels
in glial satellite cells causes down regulation
of P2X3 in nociceptive neurons, leading to
reduced pain12. In cultured hippocampal neurons
P2X7 channels are expressed in growth cones
and their inhibition promotes axon growth
(Figure 5)13. The expression of P2X7 in nerve
endings in neuromuscular junctions was nicely
demonstrated using Anti-P2X7 antibody and
immunohistochemical and electron microscopy
analysis (Figure 6)20. In a similar manner, the
channel expression in the retina was evident
in horizontal cells and was co-localized with
synaptic markers21.
Figure 5. Functional P2X7 Receptor Channel is Restricted to the Distal Region of the
Axon and Growth Cones.
A) Hippocampal neurons cultured for 3 DIV were stained with antibodies against
tyrosinated α-tubulin and P2X7 (Using Anti-P2X7 antibody, (#APR-004 or #APR-008)).
Higher-magnification views of the boxed areas show the distal region of the axon stained
for tyrosinated α-tubulin or P2X7 receptor. Scale bar: 50 µm. B) Distal region of an axon
stained with anti-α-tubulin and Anti-P2X7 antibodies. Note that α-tubulin staining, unlike
that of tyrosinated α-tubulin, does not display an increasing distal gradient. C) and D)
Graphs representing the fluorescence intensity of tubulin (red) and P2X7 (green) along
the axon in the neurons shown in A (C) and B (D), quantified using the ImageJ program. E)
Images of the most distal region of the axon and the growth cone of hippocampal neurons
stained with anti-tyrosinated-α-tubulin and Anti-P2X7 antibodies. Note the absence of
P2X7 staining in axons running parallel to a P2X7-positive distal region of an axon, where
Figure 4. Localization of Purinergic Subunits
P2X7 is located in the microtubule domain of the axon and in the actin-rich domain (inset).
Adapted from reference 13 with permission of The Company of Biologists.
in Glossopharyngeal Nerve (GPN) Neurons in
situ by Confocal Immunofluorescence.
Figure 6. Immunoreactivity for P2X7 Receptor Subunit is Present on Presynaptic Motor Nerve
Terminals from Birth Through into Adulthood.
Proximal neurons located at the bifurcation of the GPN
and CSN expressed both P2X2 (A; red, detected with
Preparations of flexor digitorum brevis muscle and tibial nerve were fixed for either immunofluorescence, immunoelectron
Anti-P2X2 antibody (#APR-003)) and P2X3 (B; green)
or confocal microscopy. Preparations of NMJs double labeled with antibodies against NF165 and SV2, visualised with FITC-
immunofluorescence; note colocalization in merged
conjugated secondary antibody (a, d and g), which labels motor axons and terminals, and P2X7 (using Anti-P2X7 antibody
images. C, Similarly, many neurons at the distal bifurcation
(#APR-004)), visualised with a TRITC-conjugated secondary antibody (b, e and h), illustrates that P2X7RS appears to be
coexpressed P2X2 and P2X3 subunits (D–F). G–I show
localised to motor nerve terminal from birth (0 day old, a–c) through 4 (d–f ) and 7 days old (g–i). Electron microscopy of a
colocalization of P2X4 purinergic subunit (green, detected
single nerve terminal bouton confirmed that immunoreactivity for P2X7RS was confined to the presynaptic nerve terminal
with Anti-P2X4 antibody (#APR-002)) with the neuronal
bouton in adults (j) and was not found on terminal Schwann cells or postsynaptic muscle fibres. A single slice taken from a
marker NF (red) in proximal GPN neurons. In J–L, there is
confocal stack through a portion of an NMJ, double labeled for P2X7RS endodomain (green, Anti-P2X7 antibody) and P2X7
colocalization of P2X4 (red) and P2X3 (green) subunits in the
ectodomain (red) antibodies (k). Extended orthogonal projections through the confocal stack (l and m) confirm co-localisation
distal population of GPN neurons. Scale bars: A–F, 50 μm;
and suggests a small spatial differentiation between the endodomain (central) and the ectodomain (peripheral) labelling, as
G–L, 100 μm.
predicted from the intra- and extracellular domains of the receptor, which these antibodies were raised against. Scale bars: a–i
Adapted from reference 23 with permission of The Society for
=10 μm, j = 500 nm, k = 5 μm.
Neuroscience.
Adapted from reference 20 with permission of Elsevier.
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Modulator No.23 Fall 2009 www.alomone.com
P2X3 in Rat Dorsal Root Ganglion.
B
A
B
C
Western blot analysis of human platelets lysate:
1. Anti-P2X1 antibody (#APR-001) (1:200).
2. Anti-P2X1 antibody, preincubated with the control
DRG
peptide antigen.
C
Spinal cord
References:
Staining of P2X3 in rat dorsal root ganglion (DRG) with Anti-P2X3 antibody (#APR-016). Cells within the DRG were stained
(see solid line frame enlarged in B) as well as fibers and the area of entry of dorsal root into spinal cord (see dashed line
frame enlarged in C). The counterstain in B and C is DAPI, a fluorescent dye visualized in the UV range.
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7. Bo, X., et al. (2003) Cell Tissue Res. 313, 159.
8. Wu, P. Y., et al. (2009) Cellular Signaling 21, 881.
Immunocytochemistry of P2X7.
Expression of P2X4 in Rat Brain.
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11. Taylor, S. R. J., et al. (2009) J. Leuk. Biol. 85, 978.
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A
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15. Fodor, J., et al. (2009) Cell Calcium 45, 421.
16. Cavaliere, F., et al. (2003) Neuroscience 120, 85.
17. Sim, J. A., et al. (2006) J. Neurosci. 26, 9006.
18. Hovarth, R. J., et al. (2009) J. Neurosci. 29, 998.
19. Vial, C. and Evans, R. J. (2000) Br. J. Pharmacol. 131, 1489.
20. Moores, T. S., et al. (2005) Brain Res. 1034, 40.
C
21. Puthussery, T., et al. (2006) Eur. J. Neurosci. 24, 7.
22. Brouns, I., et al. (2009) Histochem. Cell Biol. 131, 55.
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24. Tsuda, M., et al. (2003) Nature 424, 778.
25. Simonetti, M., et al. (2008) J. Biol. Chem. 283, 18743.
Immunocytochemistry of K562 living cells stained
with Anti-P2X7 (extracellular)-FITC antibody
(#APR-008-F).
Related Products
Compound
Flow Cytometry Analysis of Intact living
Jurkat T-cells.
Immunohistochemical staining of P2X4 in rat brain red
nucleus using Anti-P2X4 antibody (#APR-002).
A, P2X4 (green) appears in fibers surrounding cell
shapes (arrows). B, Calbindin 28K (red) appears in large
neurons. C, merge of P2X4 and Calbindin 28K suggest
variable density of P2X4 expressing fibers on red nucleus
neurons. DAPI is used as the counterstain (blue).
Product #
Antibodies to Purinergic (P2X) Receptors
Anti-P2X1_ ____________________________________
Anti-P2X2_ ____________________________________
Anti-P2X3_ ____________________________________
Anti-P2X4_ ____________________________________
Anti-P2X5_ ____________________________________
Anti-P2X6_ ____________________________________
Anti-P2X7-extracellular_ ________________________
Anti-P2X7-extracellular-FITC_____________________
Anti-P2X7_ ____________________________________
Anti-P2X7-ATTO-550____________________________
APR-001
APR-003
APR-016
APR-002
APR-005
APR-013
APR-008
APR-008-F
APR-004
APR-004-AO
Antibodies to Purinergic (P2Y) Receptors
Western blot analysis of rat brain
membranes:
1. Anti-P2X4 antibody (#APR-002),
Unstained cells.
(1:200).
Anti-P2X7 (extracellular)-FITC antibody (#APR-008-F)
2. Anti-P2X4 antibody preincubated
(10mg per 1x106 cells).
Modulator No.23 Fall 2009 www.alomone.com
with the control peptide antigen.
Anti-P2Y1______________________________________
Anti-P2Y2______________________________________
Anti-P2Y4______________________________________
Anti-P2Y6______________________________________
Anti-P2Y11____________________________________
Anti-P2Y12_ ___________________________________
Anti-P2Y13_ ___________________________________
Anti-P2Y14 (extracellular)_ ______________________
APR-009
APR-010
APR-006
APR-011
APR-015
APR-012
APR-017
APR-018
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