Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3684-3688, April 1996 Neurobiology P2X4: An ATP-activated ionotropic receptor cloned from rat brain (purinergic receptor) FLORENTINA SOTO*t, MIGUEL GARCIA-GUZMAN*t, JUAN MANUEL GOMEZ-HERNANDEZ, MICHAEL HOLLMANNt, CHRISTINE KARSCHIN, AND WALTER STUHMER*§ Department of Molecular Biology of Neuronal Signals and tGlutamate Receptor Laboratory, Max Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 G6ttingen, Germany Communicated by Step/len Heinemann, The Salk Institute for Biological Studies, La Jolla, CA, December 26, 1995 (received for review November 15, 1995)

ABSTRACT Extracellular ATP exerts pronounced biolog- remained elusive as none of the identified proteins have been ical actions in virtually every organ or tissue that has been shown to be expressed in central neurons. Here, we report the studied. In the central and peripheral nervous system, ATP cloning of a P2X receptor (P2X4) from rat brain that is acts as a fast excitatory transmitter in certain synaptic expressed in central nervous system neurons and blood vessels. pathways [Evans, R. J., Derkach, V. & Surprenant, A. (1992) The channel activated by extracellular ATP is highly Ca2+- Nature (London) 357, 503-505; Edwards, F. A., Gigg, A. J. & permeable and shows a new pharmacological profile in that Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, suramin and a,/3-methylene-ATP (a,t3meATP) are very inef- we report the cloning and characterization of complementary ficient as antagonist and agonist, respectively. DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP chan- MATERIALS AND METHODS nels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly per- Cloning of the P2X4 cDNA. Two inosine-containing degen- meable to Ca2+ and whose sensitivity is modulated by extra- erate primers based on the sequences of P2X1 (vas deferens) cellular Zn2+. Surprisingly, the current elicited by ATP is and P2X2 (PC12) were used to PCR-analyze rat brain first- almost insensitive to the common P2X antagonist suramin. In strand cDNA. The forward primer was: 5'-GTITGGGAT- situ hybridization reveals the expression of P2X4 mRNA in GTIG(A or C)IGAITATGT-3' (coding for Val-Trp-Asp-Val- central nervous system neurons. Northern blot and reverse Glu-Glu-Tyr). The reverse primer was: 5'-GTIGGIATIAT(A transcription-PCR (RT-PCR) analysis demonstrate a wide or G)TC(A or G)AA(C or T)TTICCIGC-3' (coding for distribution of P2X4 transcripts in various tissues, including Ala-Gly-Lys-Phe-Asp-Ile-Ile-Pro). The PCR thermal profile blood vessels and leukocytes. This suggests that the P2X4 consisted of 5 min at 94°C followed by five cycles of 1 min each receptor might mediate not only ATP-dependent synaptic at 94°C, 45°C, and 72°C followed by 30 cycles of 1 min each at transmission in the central nervous system but also a wide 94°C, 50°C, and 72°C. A fragment of the predicted size (-750 repertoire of biological responses in diverse tissues. bp) was isolated from a 1% agarose gel, cloned into pBluescript vector, and sequenced. The deduced amino acid sequence of was to It is now that ATP acts as a the amplified DNA homologous the previously pub- widely accepted ubiquitous lished P2X This PCR was used then as a extracellular messenger binding to a large family of membrane receptors. fragment the P2 These can probe to screen a rat brain cDNA library (Clontech) following proteins: receptors (1). purinergic receptors conventional be functionally classified in at least five subtypes: P2X, P2Y, protocols (11). and P2Z In the case of P2X ATP in Electrophysiological Characterization. Full-length coding P2U, P2T, (2). receptors, were into a vector con- the millisecond channels nonselective for mono- sequences for P2X4 cloned pSGEM, range opens from a valent cations. Certain ATP are also to structed pGEMHE (12) by inserting pBluescript receptors permeable RNA was transcribed in Ca2+ to some extent. This kind of fast ATP-mediated polylinker. Capped complementary response vitro by using Nhe I-linearized DNA. Oocyte isolation and has been identified in many tissues, including several synaptic and two-electrode of the and central nervous system, smooth handling electrophysiological recordings pathways peripheric were performed by standard techniques (13). The antagonist muscle from different organs, blood vessels, and endocrine acid see refs. A classi- pyridoxal phosphate 6-azophenyl-2',4'-disulfonic glands (for reviews, 1-5). pharmacological was for 8 min before fication of these four main groups has (PPADS) perfused agonist application. receptors comprising Suramin was purchased from Calbiochem; PPADS, from RBI; been proposed (6), suggesting molecular diversity for these and and from Three P2X have been cloned from rat vas basilen, cibacron, bromophenol blue, Sigma. proteins. receptors To estimate Ca2+ permeabilities, oocytes were injected with deferens smooth muscle (P2Xi) (7), PC12 cells (P2X2) (8), and 50 nl of 200 mM EGTA to 8.0 with 20 rat dorsal root These P2X (adjusted pH NaOH) ganglia (P2X3) (9, 10), respectively. min prior to recording, preventing Ca2+ from activating en- receptors show pharmacological as well as kinetic (degree of Cl- channels. Glutamate to dogenous Ca2+-dependent receptor desensitization) differences. The capacity form heteromul- clones included a heteromeric combination of timeric complexes further increases the diversity of ATP- N-methyl-D- activated currents. For example, the P2X2 and P2X3 forms have been shown to associate in with Abbreviations: PPADS, pyridoxal phosphate 6-azophenyl-2',4'- multimers, pharmaco- disulfonic acid; a,q3meATP, a,3-methylene-ATP; NMDA, N-methyl- logical and kinetic properties different from the homo- D-aspartate; KA, kainate; AMPA, D,L-a-amino-3-hydroxy-5-methyl- oligomeric channels (10). 4-isoxazole-propionic acid; NMG, N-methyl-D-glucamine; RT, reverse Despite the molecular characterization of several ATP transcription; Ercv, reversal potential; 2meSATP, 2-methylthio-ATP; receptors, the central nervous system P2X receptor(s) have TFR, transferrin receptor; [Zn2+]o and [Ca2+]o, extracellular con- centrations of Zn2+ and Ca2+. *F.S. and M.G.-G. contributed equally to this paper. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. X93565). 3684 Downloaded by guest on September 23, 2021 Neurobiology: Soto et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3685

P2xl (V. D. MARRLQDELSAFFFE DETPRMVLVRNKKVGVIFLIQLVVL\IGVFVYE SSDL-SCVSVKLGLAVTQLQGLGPQVWDVA AHGDSS 99 P2x2 (PC12) MVRRLARGCWSAFWDYETPKVIVVRNRRLGFVHRMVQLLILLYFVWYVFIVQKSYCDSETGPE SSIIT VKGKITMSED -----KVWDVEEYVKPEGGSV 95 P2x3 (DRG) MNCISDFFTYETTKSVVVKSWTICIINRAVQLLIISYFVGWVFLHEKAY VRDTAIESSVVT VKGFGRYAN -----RVMDVSEYVTFPQGTSV 89 P2x4 (Brain) MA-GCCSVLGSFLF E[DjTPRIVLIRSRKVJLMNjAV1QLILA[YVIGWFVWE GYETDSVV- _IVTTASGCVAVTNTSQLGFRIWADAYIPFAQEENS 98 Ml Y P2xl (V. D. ) FVVMfNFICTPQQTQGH AN-PE-GG1}QDCSGTPTKAERKAEQIR NCPF-NGTVKTCEIFGWCPVVDDKIPSPALLREAENFFLFIISi1SFP 196 P2x2 (PC12) VSI ITRIEVTPSTLGTCPESMRVHSSTMHSEDDCIA3QLDMQGNI RTGHCVPYYHGDSKTCEVSAWCPVEDGT-SDNHFLGKMAPNFTILIKNISIHYP 194 P2x3 (DRG) FVIITKIIVTENQMQGFCPEN--EEKYRCVSESQC--PERFPGGILGRI CVNY-SSVLRTCEIQGWCPEV DT-VEMPIM -MEAENFTIFIKNSIRFP 182 P2x4 (Brain) LFIM NMIVTVNQTQSTCPEI-PDKTSICNSEADCJTPSVDTHSSVATGRCVPF-NESVKCEVAAWCPVENDVGVPTPAFLKAAENLLTVKNNIWYP 196 Y Y Y P2x1 (V.D.) RFKVNRRELVEEVNGTYMRKLYHKIQFH ILVFRES RSLREEVV K^I HVRH IHG - EKNLFNFRF 293 P2x2 (PC12) KFKFSKGNIASQKSDY-LKHCTFDQDS PYCPIFRLfFIVEKA ENFTELAHK|GGVIVINNCI DLLSESECNP S FRRDPKYD--PASSNFRF 291 P2x3 (DRG) LFNFEKGNLLPNLTDKDIKRCRFHPEKAPFCPILRVfDVVKFAQDFAKLARTGGVLIKIGI VCDLKAWDQCIPKY SFT RDGVSEKSSVS FR 282 P2x4 (Brain) KFNFSKRNILPNITTSYLKSCIYNAQT PFCPIFRLjTIVEDAHSFQEMAVE GIMIQIK RAAS SRRDTRDLEHNV FR 296 Y Y P2xl (V. D. ) ARHFVQ-NGTNR FKFHI D GEFGKFIPFLTTIGSGIGIFATVLOLLLHILPKRHYYKQKKFKEYAEDMGPGE-GEHDPVATSSTLG 391 P2x2 (PC12) HAKYYKINGTTTTRTLIKAYIRIV IVHGQAGKFS LIPTIINLATALTSIGVGSFLC DWI LLTFMNKNKLYSHKKF DKVRTPKHPSSRWPVTLALVLGQI 391 P2x3 (DRG) HAKYYKMENGSEYRLLKAFG IRFEVLVYAGKFNIIPTIISSVAAFTSV|GVGTVLCI I LNFLKGADHYKARKFEEVTETTLKGTASTNPVFASDQAT 382 P2x4 (Brain) AKYYRDLAGKEQRLKAYIRFEI IVFKAGKFDIIPTMINVGSGLALLVATVLCEVIVLYCMKKKYYYRDKKYKEYVEDYEQGLSGEMNQ 388 M2 P2xl (V.D.) LQENMRTS 399 P2x2 (PC12) PPPPSHYSQDQPPSPPSGEGPTLGEGAELPLAVQSPRPCSISALTEQVVDTLGQHMGQRPPVPEPSQQDSTSTDPKGLAQL 472 P2x3 (DRG) VEKQSTDSGAYSIGH 397 FIG. 1. Multiple aligment of the P2X amino acid sequences. Residues conserved in all four rat sequences are boxed. The putative transmembrane segments are underlined. The Y symbols indicate P2X4 N-glycosylation sites. The original tissues from which the different receptors were cloned are indicated in parentheses. V.D., vas deferens; DRG, dorsal root ganglia. aspartate (NMDA) receptor subunits (NMDARi-la and TACCCAGGACGACTTTATC-3' (reverse) (PCR fragment NR2B) (14), the kainate (KA) receptor subunit GluR6(Q) length, 281 bp). The blot was hybridized at high stringency with (15), and the D,L-a-amino-3-hydroxy-5-methyl-4-isoxazole- appropriate [a-32P]dCTP random-primed probes for the TFR propionic acid (AMPA) receptor heteromeric subunit combi- and P2X4 genes. An equivalent result was obtained with an nation GluRl plus GluR2 (16). For quantitative evaluations, antisense primer to the 3' untranslated region of the P2X4 gene the Goldman-Hodgkin-Katz (GHK) equation modified to (data not shown). include divalent cations (17) was used. N-Methyl-D-glucamine In Situ Hybridization. Digoxigenin (DIG)-labeled cRNA (NMG) was impermeable as evidenced by the absence of a probes were generated by in vitro transcription with DIG-UTP shift of the reversal potential upon increasing NMG concen- (Boehringer Mannheim). The cDNA template was the 750-bp tration. The ratio of Ca'2 to monovalent-cation P2X4 PCR fragment. Adult rat brain and spinal cord sagittal published sections (10-16 ,tm) were cut on a cryostat, fixed, and dehy- permeability (Pc-/Pmono) of 1.2 for the KA receptor GluR6(Q) and immu- and the measured reversal of drated. Pretreatments, hybridization of sections, (15) potential (Erev) GluR6(Q) nological detection were carried out as reported (20). Color of -56.5 + 3.7 mV = in 8 mM solution (n 3) Ca2+/NMG development was stopped after 24-30 hr. Hybridizations of allowed an estimate of the activity of monovalent cations under sense cRNA probe to adjacent control sections were negative our experimental conditions. Pca,/Pmono values for the other (data not shown). receptors were then calculated from their Ercv values in 8 mM Ca2+/NMG [-11.7 ± 1.7 (n = 3), -29.4 + 1.0 (n = 4), and -118.8 ± 1.1 (n = 3) (in mV) for the NMDA, P2X4, and RESULTS AND DISCUSSION AMPA receptors, respectively]. Ca2+ activities were used (18), Cloning, Sequence, and Structure of a P2X Receptor from and Er,v values were corrected for liquid junction potentials. Rat Brain. By using degenerate oligonucleotides based on RNA (Northern) Blot and Reverse Transcription-PCR (RT- P2X1 and P2X2 sequences, we generated a specific probe to PCR). Total RNA was prepared by the single-step method screen a rat brain cDNA library and isolated a cDNA fragment (19). Poly(A)+ RNA was purified by using oligo(dT)25- that shows homology to the previously cloned ATP receptors. magnetic beads (Dynal, Promega), separated in a denaturing P2X4 shares 50% amino acid identity with the rat vas deferens formaldehyde-agarose gel [-3 ,ig of poly(A)+ RNA for brain, (P2Xi) (7), 44% with the PC12 (P2X2) (8), and 44% with the heart, lung, uterus, testis, kidney, small intestine, and adrenal rat dorsal root ganglion (P2X3) receptors (9, 10). The homol- and -1 utg for other tissues], capillary-transferred to nylon ogy between the four rat channels is evenly distributed along membranes, and UV-crosslinked. The membrane was hybrid- the sequence (28% identity), except in the C-terminal part, ized at high stringency with a [a-32P]dCTP random-primed suggesting a common pattern for the three-dimensional struc- DNA probe (Xba I-BamHI fragment comprising the first 850 ture of the protein. The P2X4 receptor contains an open bp of P2X4). Single-tube RT-PCR was performed in 50-dl reading frame of 1164 nucleotides encoding a 388-amino acid reaction volumes containing 500 ng of total RNA, 1 ,LM each protein with a predicted molecular mass of 43 kDa. The of of and transferrin receptor principal features of other members of the P2X family are pairs P2X4- (TFR)-specific present in the new receptor, including two highly hydrophobic primers (see below), 200 ,LM dNTPs, 40 units RNAsin, 2 units and The has six virus reverse 2.5 units of regions (Ml M2, Fig. 1). P2X4 protein of avian myeloblastosis transcriptase, consensus sites for in with DNA and 1 x DNA reaction N-glycosylation (Y symbols Fig. 1), Taq polymerase, Taq polymerase only one being conserved between the four receptors (Asn- buffer (Promega). Incubation at 50°C for 15 min was followed 184). There are 11 cysteine residues conserved in all cloned by a PCR thermal profile of 5 min at 94°C and 35 cycles of 40 P2X channels, suggesting an important role for their tertiary s each at 940C, 57°C, and 72°C. Every PCR was also performed structure (5). An ATP-binding motif [P-loop, Gly-Xaa4-Gly- in the absence of reverse transcriptase (see Fig. 3B) to check Lys-(Thr or Ser); ref. 21] is absent in the P2X receptors. for genomic DNA contamination of the corresponding RNA. Nevertheless, a similar sequence, Gly-Xaa-Ala-Gly-Lys-Phe, The P2X4 primers were 5'-CGTGGCGGACTATGTGATT-3' just preceding the M2 segment, is conserved in all of them (8). (forward) and 5'-GTGATGTTGGGGAGGATGTTC-3' (re- Functional Expression of P2X4 Confirms That It Encodes verse) (PCR fragment length, 375 bp). The TFR primers were an ATP-Gated Channel with High Ca2+ Permeability. To 5'-GGAACCAGACCGCTACATT-3' (forward) and 5'- characterize the functional properties of the P2X4 receptor, we Downloaded by guest on September 23, 2021 3686 NerbooySooeal Proc. Natl. Acad. Sci. USA 93 (1996)

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. 0 1 2 3 0 00 2.34367 0-cr^ xt/ -808-120- *U I10- 1 100 102 10 7 Log[Agonist], (gM) Log(aca) (mM) FIG. 2. Electrophysiological and pharmacological properties of P2X4 receptors expressed in Xenopus laevis oocytes. (A) P2X4 inward currents elicited by 100 gM of the following agonists: ATP, 2-methylthio-ATP (2meSATP), CTP, a,pmeATP, and dATP (holding potential, -70 mV). The interval between all applications was 3 min to allow for full recovery from desensitization. (B) Dose-response curves of P2X4 for ATP (0), 2meSATP (o), CTP (X), and ATP in the presence of 10 ,iM extracellular zinc concentration ([Zn2+].) (0) (n . 4). Currents are normalized to that elicited by 20 jjiM ATP without [Zn2+] in the same cell. Continuous lines are Boltzmann fits to the data except for 2meSATP. (C) Current-voltage relationship of P2X4 (50 ,uM ATP, normalized to the current at - 120mV; n = 3). (D) Ca2+ activity (aca)-dependent shifts of the reversal potential in NMDA (0), P2X4 (*), KA (o), and AMPA (m) receptors. The AMPA receptor is virtually Ca2+-impermeable. The extracellular solution in A is 115 mM NaCl/2.5 mM KCl/1.8 mM CaCl2/10 mM Hepes, pH 7.2; the extracellular solution in B and C is as above but with MgCl2 replacing CaCl2 to avoid Ca2+-activated Cl- currents. Solutions used in D to estimate Ca2+ permeabilities contained 10 mM Hepes; 2, 4, or 8 mM CaCl2; and 117.2, 114.2, or 108.2 mM NMG, respectively (pH 7.2).

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FIG. 3. Tissue distribution of P2X4transcripts. (A) Northern blot assay of P2X4 transcript distribution in adult rat tissues. The sizes (in kilobases) of the molecular mass markers are from an RNA ladder (Boehringer Mannheim). (B) Analysis of P2X4 mRNA tissue expression by RT-PCR and Southern blot autoradiography with a P2X4-specific probe (P2x arrow) and a rat TFR probe (TFR arrow) as a control for RT-PCR efficiency. The presence (+) or absence (-) of reverse transcriptase is indicated at the top of the lanes; H20 indicates control samples without RNA. Downloaded by guest on September 23, 2021 Neurobiology: Soto et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3687 used the Xenopus oocyte expression system. Only in comple- P2X blocker PPADS (25). Thus, 100 p/M PPADS only reduces mentary RNA-injected oocytes did ATP evoke a rapid inward the current elicited by 5 p.M ATP to 90.6% ± 13.5 of the control current that desensitized in the continuous presence of the (n = 3). However, other P2X blockers such as the two isomers of agonist (Fig. 2A). The P2X4 receptor is activated by nucleotide reactive blue, basilen blue and cibacron blue, at 100 ,uM were able analogues with the following order of efficacy at 100 uM: ATP to reversibly reduce the responses of 5 ,uM ATP by 74.6% + 4.2 > 2-methylthio-ATP (2meSATP) - CTP > a,/3-meATP > (IC50 - 50 ,pM) and 44.0% ± 2.9 (IC50 - 120 ,pM) (n = 4), dATP. No significant responses (<1% of ATP current) were respectively. detected with 100 /AM of ADP, AMP, 3,y-methylene-ATP, The low sensitivity to 2meSATP (Fig. 2B) suggests that P2X4 GTP, and adenosine. The neurotransmitter receptor agonists could be classified pharmacologically within the third P2X- (at 100 ,aM) acetylcholine (with 50 ,iM atropine), nicotine, purinoceptor subclass (6). This group is represented by the glutamate, y-aminobutyric acid (GABA), glycine, and seroto- receptor expressed in the vascular smooth muscle (saphenous nin did not elicit currents. Dose-response analysis gave half- artery). Nevertheless, the agonist order of potency determined maximal effective concentrations (EC5o) of 6.9 ± 0.8 /aM for in tissues may be equivocal because of different ecto-ATPase ATP, with a Hill coefficient (nH) of 1.1 ± 0.1 (Fig. 2B). activities (26). The suramin-insensitive behavior of the P2X4 Extracellular zinc concentration, [Zn2+]o, has been reported to receptor contrasts with all of the previously reported ATP increase the ATP sensitivity of both native (22) and cloned activated currents in vivo. Therefore, the cloning of P2X4 P2X receptors (8). Dose-response curves in the presence of 10 expands the list of criteria for further classifications of the P2X liM [Zn2+]. showed an increase in agonist sensitivity (EC5o = receptors and for the pharmacological identification of new 2.5 + 0.4 J,M, nH = 1.4 + 0.2), without alteration of the subtypes of ATP-activated currents in vivo. maximal response (Fig. 2B). This modulation by Zn2+ of the The current-voltage relationship displayed an inward rec- ATP channels might increase excitability in certain regions of the tification (Fig. 2C) as observed for other P2X receptors (7-10, brain. For example, Zn2+ is present at high concentrations in the 27). P2X4 is almost equally permeable to Na+ and K+ as mossy fiber terminals of the hippocampal formation, from where revealed by shifts of the Erev in buffers containing various it can be released upon stimulation (23). The classical P2X concentrations of these ions. Erev did not change when external antagonist suramin (6) turned out to be a very inefficient blocker Cl- was partially replaced by methane sulphonate, indicating of P2X4 compared with its reported actions on other cloned and Cl- impermeability (data not shown). To investigate the Ca2+ native P2X receptors (5, 10, 24). Notably, in the presence of 5 JuM permeability of P2X4, we analyzed the Ercv in buffers contain- ATP, 100 ,LM suramin merely gives a 20.1% + 3.2 block of the ing low concentrations of extracellular Ca2+, [Ca2+]o, as the current (n = 4). Further increase of suramin concentration to 500 sole permeable ion. With increasing [Ca2+], Erev shifted /iM (n = 3) blocks the ATP current only by 33.4 ± 6.1% (n = 3). toward positive potentials, indicating Ca2+ permeability (Fig. There was no clear difference in the action of suramin and the 2D). To gauge the magnitude of the observed Ca2+ perme- A

FIG. 4. Cellular localization of P2X4 mRNA as revealed by in situ hybridization. Positive hybridization signals appear dark in bright-field photomicrographs of rat brain and spinal cord sections. (A) Hippocampus. Dentate gyrus granule cells and CA1 /CA3 pyramidal cells are moderately labeled. (B) Cerebellar cortex. Purkinje cells are more strongly labeled than granular cells. (C) Pontine nucleus. Numerous neurons are positive. (D) Spinal cord. Large motoneurons are heavily labeled. [Bars = 250 jum (A) and 100 ,Am (B-D).] Downloaded by guest on September 23, 2021 3688 Nuoilgy ooe l Proc. Natl. Acad. Sci. USA 93 (1996) ability, we compared P2X4 to various subtypes of recombinant Note Added in Proof. After submission of this manuscript, three rat glutamate receptors of known Ca2+ permeabilities: a cDNAs were reported (33-35) whose sequences are identical to the heteromeric NMDA receptor, a KA receptor, and a hetero- P2X4 reported here. meric AMPA receptor, serving as benchmarks for ligand-gated ion channels with high, intermediate, and very low Ca2+ We thank Dr. Jim Boulter, Salk Institute, San Diego, for kindly The relative shifts of providing the NR2B clone; the late Dr. Peter Hess, Harvard Medical permeability, respectively (Fig. 2D). Ercv School, Boston, for pGEMHE; Drs. Robert J. Chow and Synnove Beckh observed at various [Ca2+] indicate P2X4 has a Ca2- perme- for critically reading the manuscript; and Katja Anttonen for excellent ability slightly lower than that of the NMDA receptor, but technical assistance. M.H. is a Heisenberg Fellow of the Deutsche significantly higher than that of the KA receptor (Fig. 2D). A Forschungsgemeinschaft. Financial support was obtained from the Max- more quantitative estimate of the Ca2+ permeability of P2X4 Planck Gesellschaft. was obtained by using a modified Goldman-Hodgkin-Katz equation. We found Pca/Pmono ratios of 10.4 and 0.09 for 1. Dubyak, G. R. & El-Moatassim, C. (1993) Am. J. Physiol. 265, NMDA and AMPA receptors, respectively, which is close to C577-C606. the values of 10.6 and 0.05-0.14 reported earlier (28, 29). P2X4 2. Chen, Z. P., Levy, A. & Lightman, S. L. (1995) J. Neuroendocri- has a PCa/Pmono value of 4.2. The P2X4 represents the first ATP nol. 7, 83-96. in central nervous neurons with a 3. Bean, B. P. (1992) Trends Pharmacol. Sci. 13, 87-90. receptor expressed system 4. Edwards, F. A. & Gibb, A. J. (1993) FEBS Lett. 325, 86-89. demonstrated high Ca2+ permeability, since the vas deferens 5. Surprenant, A., Buell, G. & North, R. A. (1995) Trends Neurosci. (PCa/PNa = 4.8) (7) and dorsal root ganglion forms (PCa/PNa 18, 224-229. = 4) (10) are not present in brain. 6. Abbracchio, M. P. & Burnstock, G. (1994) Pharmacol. Ther. 64, Tissue Distribution Analysis Shows P2X4 mRNA Expres- 445-475. sion in Brain Neurons and Many Other Tissues. Northern blot 7. Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A., assays demonstrate a wide tissue distribution of the P2X4 Surprenant, A. & Buell, G. (1994) Nature (London) 371,516-519. transcript. Indeed, a strong band of -2.4 kb is detected not 8. Brake, A. J., Wagenbach, M. J. & Julius, D. (1994) Nature only in brain but in all tissues analyzed except testis, in which (London) 371, 519-523. a faint band was observed The 9. Chen, C. C., Akopian, A. N., Sivilotti, L., Colquhoun, D., Burn- only very (Fig. 3A). P2X4 J. N. Nature mRNA was confirmed and extended stock, G. & Wood, (1995) (London) 377, 428-431. expression pattern (Fig. 10. Lewis, C., Neidhart, S., Holy, C., North, R. A., Buell, G. & 3B) by RT-PCR. The detection of P2X4 mRNA in all tissues Surprenant, A. (1995) Nature (London) 377, 432-435. analyzed suggests that this receptor could be expressed in a 11. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular common cell type present in all organs. Previous work strongly Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, indicates the presence of P2X receptors in smooth muscle of Plainview, NY), 2nd Ed. blood vessels, including large-, medium- and small-sized ar- 12. Liman, E. R., Tytgat, J. & Hess, P. (1992) Neuron 9, 861-871. teries (30, 31). The detection of P2X4 transcripts in ascen- 13. Stuhmer, W. (1992) Methods Enzymol. 207, 319-345. dant aorta"and vena cava (Fig. 3B) suggests that P2X4 is 14. Hollmann, M., Boulter, J., Maron, C., Beasley, L., Sullivan, J., presumably present in the vascular system of most organs and Pecht, G. & Heinemann, S. (1993) Neuron 10, 943-954. therefore an role in the modulation of 15. Egebjerg, J. & Heinemann, S. F. (1993) Proc. Natl. Acad. Sci. might play important USA 90, 755-759. blood vessel contractility. P2X4 mRNA was also detected in 16. Hollmann, M., Hartley, M. & Heinemann, S. (1991) Science 252, rat blood leukocytes (Fig. 3B). Although the function of 851-853. extracellular ATP in immune responses is unknown, exten- 17. lino, M., Ozawa, S. & Tsuzuki, K. (1990)J. Physiol. (London) 424, sive reports show that ATP is involved in the activation and 151-165. modulation of immunological responses (1, 32). The P2X4 18. Shatkay, A. (1968) Biophys. J. 8, 912-919. mRNA expression in these specialized cells opens the pos- 19. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. sibility to analyze the role of this protein in ATP-evoked 20. Bartsch, S., Bartsch, U., Dorries, U., Faissner, A., Weller, A., DNA synthesis of lymphocytes, blastogenesis, cell-mediated Ekblom, P. & Schachner, M. (1992) J. Neurosci. 12, 736-749. and 21. Saraste, M., Sibbald, P. R. & Wittinghofer, A. (1990) Trends killing, apoptosis. 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Kennedy, C. & Leff, P. (1995) Trends Pharmacol. Sci. 10, 168-174. in and olive and in 27. Nakazawa, K., Inoue, K., Fujimori, K. & Takanaka, A. (1991) (Fig. 4C); vestibular, reticular, inferior nuclei; Pflugers Arch. 418, 214-219. motoneurons of the spinal cord (Fig. 4D). Lower levels of P2X4 28. Mayer, M. L. & Westbrook, G. L. (1987)J. Physiol. (London) 394, mRNA expression is observed in the cortex, olfactory bulb, 501-527. thalamus, and hypothalamus (data not shown). 29. Burnashev, N., Zhou, Z., Neher, E. & Sakmann, B. (1995) J. The pharmacological profile of P2X4 (almost insensitive to Physiol. (London) 485, 403-418. suramin and a,43meATP; low sensitivity to 2meSATP) con- 30. Benham, C. D. (1989) J. Physiol. (London) 419, 689-701. trasts with that of previously described ATP-activated chan- 31. Bo, X. & Burnstock, G. (1993) J. Vasc. Res. 30, 87-101. nels. Therefore, the cloning of P2X4 expands the criteria for 32. Naumov, A. P., Kaznacheyeva, E. V., Kiselyov, K. I., Kuryshev, further classifications of the P2X receptors and suggests spe- Y. A., Mamin, A. G. & Mozhayeva, G. N. (1995) J. Physiol. cific tests for the identification of new sub- (London) 486, 323-337. pharmacological 33. Bo, X., Zhang, Y., Nassar, M., Burnstock, G. & Schoepfer, R. types of ATP-activated currents in vivo. The wide distribution (1995) FEBS Lett. 375, 129-133. in the central nervous system and in blood vessels together with 34. Buell, G., Lewis, C., Collo, G., North, R. A. & Surprenant, A. the high Ca2+ permeability of the P2X4 receptor suggest that (1996) EMBO J. 15, 55-62. new physiological roles for the ATP-activated currents remain 35. Seguela, P., Haghighi, A., Soghomonian, J. J. & Cooper, E. to be discovered. (1996) J. Neurosci. 16, 448-455. Downloaded by guest on September 23, 2021