The P2Y nucleotide receptors in the human genome.

Since the first identification of P2Y receptor sequences in 1993, it has quickly become apparent that this family of the G-protein coupled receptors is very diverse. Members of this receptor family are activated extra-cellularly by a wide variety of adenosine and uridine nucleotides including sugar-nucleotides. The recent decipherment of the Human Genome has enabled us to search for new, yet undiscovered P2Y receptor subtypes. In this article we examine the relationships of six orphan G-protein coupled receptor (GPCR) sequences which show considerable sequence homology to various P2Y receptors. The clustering at a few chromosomal loci of P2Y receptor genes and their related orphan genes further suggests that particular P2Y subsets were derived from the same ancestral gene during evolution.

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

An elusive receptor is finally caught: P2Y(12'), an important drug target in platelets.

Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y(12), that acts with the P2Y(1) receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X(1) receptor ion channel, contributes to platelet activation.

Adenosine nucleotides acting at the human P2Y1 receptor stimulate mitogen-activated protein kinases and induce apoptosis.

For the widely distributed P2Y receptors for nucleotides, the transductional and functional responses downstream of their coupling to G proteins are poorly characterized. Here we describe apoptotic induction and the associated differential stimulation of mitogen-activated protein (MAP) kinase family members by the human P2Y(1) receptor. The potent P2Y(1) receptor agonist, 2-methylthio-ADP (2-MeSADP), stimulated the extracellular-signal regulated kinases (ERK1/2) (EC(50) approximately 5 nm) as well as several, but not all isoforms detected, of the stress-activated protein kinase (SAPK) family. Phospho-isoforms of p38 were unaffected. The induced kinase activity was blocked by the P2Y(1) receptor-selective antagonist, adenosine-2'-phosphate-5'-phosphate, but unaffected by pertussis toxin. In addition, the endogenous ligand ADP, and significantly also 2-MeSATP, induced concentration-dependent phosphorylation changes in the same MAP kinase family members. The sustained activation of ERK1/2 was associated with Elk-1 phosphorylation that was abolished by the MEK1 inhibitor, PD 98059. However, the concomitant transient activation of the SAPKs was not sufficient to induce c-Jun or ATF-2 phosphorylation. The transient phase of the ERK activity was partially inhibited either by the phosphatidylinositol 3-kinase inhibitor, LY 294002, or the PKC inhibitor, Gö 6976. In addition, the Src inhibitor, PP1, or expression of dominant negative Ras also attenuated the transient phase of ERK phosphorylation. In contrast, inhibition of Ras or Src had no effect on the sustained ERK activity, which was critically dependent on phosphatidylinositol 3-kinase. The transient SAPK activity was suppressed by expression of a dominant negative form of MKK4. Furthermore, this kinase-deficient mutant inhibited 2-MeSADP-induced caspase-3 stimulation and the associated decrease in cell number. In conclusion, adenosine di- and triphosphate stimulation of the human P2Y(1) receptor can transiently activate the Ras-ERK cascade via the cooperative effects of phosphatidylinositol 3-kinase, Src and PKC. The sustained ERK stimulation, via a Ras-insensitive pathway, culminates in Elk-1 activation without inducing a proliferation effect. The transient SAPK activity did not evoke transcription factor phosphorylation but was required for the P2Y(1) receptor-mediated apoptotic function.

Structural diversity despite strong evolutionary conservation in the 5'-untranslated region of the P-type dystrophin transcript.

Analysis of the 5'-flanking regions of the Purkinje (P-) dystrophin genes and mRNAs in different species revealed strong sequence conservation but functional diversity. Multiple transcription initiation sites were identified in cerebella and muscles, tissues expressing P-dystrophin. The predominant initiation site was conserved, with another muscle-specific site located upstream. Despite sequence homology, significant tissue- and species-specific structural diversity in the P-type 5'-ends exists, including alternative splicing within the 5'-untranslated region combined with alternative splicing of intron 1. One amino terminus is conserved in mammals and, to a lesser extent, in chicken. However, alternative usage of ATG codons may result in a choice of N-termini or translation of short upstream ORFs in different species. Promoter activity of a fragment upstream of the cap site was shown by transient expression in myoblasts and in vivo following intramuscular injection. It is tissue- and developmentally regulated. Analysis of promoter deletions suggests the existence of negative regulatory elements in the proximal region.

Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells.

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.

alpha(1B) adrenergic receptors in gonadotrophin-releasing hormone neurones: relation to Transport-P.

1. Peptidergic neurones accumulate amines via an unusual uptake process, designated Transport-P. [(3)H]-prazosin binds to alpha(1) adrenoceptors on these cells and is displaceable by unlabelled prazosin in concentrations up to 10(-7) M. However, at greater concentrations of prazosin, there is a paradoxical accumulation of [(3)H]-prazosin which we have attributed to Transport-P. Uptake of prazosin via Transport-P is detectable at 10(-10) M prazosin concentration, is linear up to 10(-7) M and at greater concentrations becomes non-linear. In contrast, in noradrenergic neurones, noradrenaline uptake is linear and saturates above 10(-7) M. In noradrenergic neurones and in non-neuronal cells, there is no uptake of prazosin in concentrations up to 10(-6) M, suggesting that Transport-P is a specialised function of peptidergic neurones. 2. Using a mouse peptidergic (gonadotrophin-releasing hormone, GnRH) neuronal cell line which possesses Transport-P, we have studied the interaction of alpha(1) adrenoceptors with Transport-P. Polymerase chain reactions and DNA sequencing of the products demonstrated that only the alpha(1B) sub-type of adrenoceptors is present in GnRH cells. 3. In COS cells transfected with alpha(1b) adrenoceptor cDNA and in DDT(1) MF-2 cells which express native alpha(1B) adrenoceptors, [(3)H]-prazosin was displaced by unlabelled prazosin in a normal equilibrium process, with no prazosin paradox in concentrations up to 10(-6) M. In DDT(1) MF-2 cells, [(3)H]-prazosin was displaced likewise by a series of alpha(1) adrenergic agonists, none of which increased the binding of [(3)H]-prazosin. Hence, the prazosin paradox is not due to some function of alpha(1) adrenoceptors, such as internalization of ligand-receptor complexes. 4. In neurones which possess Transport-P, transfection with alpha(1b) adrenoceptor cDNA resulted in over-expression of alpha(1B) adrenoceptors, but the prazosin paradox was unaltered. Thus, alpha(1) adrenoceptors and Transport-P mediate distinct functions in peptidergic neurones.

Imagination and reality in the search for the P2Y receptors.

A great body of evidence based on tissue and organ physiology and pharmacology led to the recognition, widespread by about 1990, that there must be cell membrane receptors for extracellular nucleotides to transduce their effects. This evidence was provided by the pioneering work of Geoffrey Burnstock and those who worked with him, or was developed by others starting from that information. This article will review how we could start from that foundation to clone the first known gene for such a receptor, P2Y(1). Some unusual properties of that receptor were revealed. I will consider further the P2Y receptors as a class - its definition, now that many such genes have become known. Imagination and reality have been intertwined in this saga.

Inhibition of potassium and calcium currents in neurones by molecularly-defined P2Y receptors.

Messenger RNAs and cDNAs for individual cloned P2Y(1), P2Y2 and P2Y(6) nucleotide receptors have been expressed by micro-injection into dissociated rat superior cervical sympathetic neurones and the effects of stimulating the expressed receptors on voltage-activated N-type Ca(2+) currents and M-type K(+) currents recorded. Both currents were reduced by stimulating all three receptors, with the following mean IC(50) values: P2Y(1) (agonist: ADP) - I(K(M)) 6.9 nM, I(Ca) 8.2 nM; P2Y(2) (agonist: UTP) - I(K(M)) 1.5 microM, I(Ca) 0.5 microM; P2Y(6) (agonist: UDP) - I(K(M)) 30 nM, I(Ca) 5.9 nM. Inhibition of I(K(M)) was voltage-independent and insensitive to Pertussis toxin; inhibition of I(Ca) showed both voltage-sensitive and insensitive, and Pertussis toxin-sensitive and insensitive components. It is concluded that these P2Y receptors can couple to more than one G protein and thereby modulate more than one ion channel. It is suggested that these effects on K(M) and Ca(N) channels may induce both postsynaptic excitory and presynaptic inhibitory responses.

The P2Y(1) receptor closes the N-type Ca(2+) channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists.

The rat P2Y(1) nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro-injection of the receptor cRNA or cDNA. ADP inhibited the N-type Ca(2+) current by 64%, with EC(50) 8.2 nM, an action blocked competitively by the P2Y(1) receptor antagonist adenosine 3', 5'-bis-phosphate (K(i) 0.7 microM). 2-Methylthio-ADP inhibited the Ca(2+) current likewise, but with EC(50) 0.57 nM, giving the highest potency reported therewith for P2Y(1). Significantly, ATP and 2-methylthio-ATP were also agonists, the latter again at a very high potency (EC(50) 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N-type Ca(2+) currents and hence can reduce transmitter release or increase neuronal excitability.

Functional characterization of the P2X(4) receptor orthologues.

1. The aim of this study was to functionally characterize the recombinant mouse P2X(4) receptor and to compare its pharmacological properties with those of the human and rat orthologues. 2. Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X(4) receptors, using Cs-aspartate containing electrodes (3 - 8 MOmega) in a HEPES-buffered extracellular medium. 3. The agonist potency of ATP at the three species orthologues was similar, with mean EC(50) values of 2.3 microM, 1.4 microM and 5.5 microM, respectively. 4. Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X(4) receptors (EC(50)=2.6 and 3.0 microM), but was significantly less potent at the rat orthologue (EC(50)=20.0 microM). alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X(4) and 24% at the human P2X(4) receptor. 5. In contrast to the other species orthologues, alpha,beta-meATP failed to elicit a significant agonist response at rat P2X(4) receptors, and was found to act as an antagonist, with an IC(50) of 4.6 microM, against 10 microM ATP. 6. Mouse P2X(4) receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC(50)=10.5 microM), as were human P2X(4) receptors (IC(50)=9.6 microM). The rat receptor however, showed a low sensitivity to PPADS (IC(50)>100 microM). 7. All three orthologues were relatively suramin-insensitive (IC(50)>100 microM) and insensitive to 1-[N, O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC(50)>3 microM). 8. Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X(4) receptor, and differ markedly from the rat receptor.

Dual effect of nucleotides on P2Y receptors.

The interaction of ADP, 2MeSADP, and ADPbetaS with the adenine nucleotide receptor P2Y1 in the hP2Y1-1321N1 cell line and of UDP with a receptor or receptors recognizing pyrimidine nucleotides in NG108-15 cells was studied over a wide range ofligand concentrations. Bell-shaped dose-response curves for stimulation of phosphoinositide hydrolysis were obtained in these cells. This dual behavior of the agonists studied was characterized by two dissociation constants, K(agon) and K(antag), which quantify the agonistic and antagonistic activity of these ligands and can be compared with the conventional EC50 and IC50 values, respectively. The data revealed a common pattern of agonistic and antagonistic behavior of nucleoside diphosphates and their derivatives at these two types of P2Y receptors, pointing to some similar properties of their nucleotide binding sites.

Dual coupling of heterologously-expressed rat P2Y6 nucleotide receptors to N-type Ca2+ and M-type K+ currents in rat sympathetic neurones.

1. The P2Y6 receptor is a uridine nucleotide-specific G protein-linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro-injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N-type Ca2+ (I(Ca(N))) and M-type K+ (I(K(M))) currents. 2. In P2Y6 cRNA-injected neurones, UDP or UTP produced a voltage-dependent inhibition of I(Ca(N)) by approximately 53% in whole-cell (disrupted-patch) mode and by 73% in perforated-patch mode; no inhibition occurred in control cells. Mean IC50 values (whole-cell) were: UDP, 5.9+/-0.3 nM; UTP, 20+/-1 nM. ATP and ADP (1 microM) had no significant effect. Pertussis toxin (PTX) substantially (approximately 60%) reduced UTP-mediated inhibition in disrupted patch mode but not in perforated-patch mode. 3. Uridine nucleotides also inhibited I(K(M)) in P2Y6 cRNA-injected cells (by up to 71% at 10 microM UTP; perforated-patch). Mean IC50 values were: UDP, 30+/-3 nM; UTP, 115+/-12 nM. ATP (10 microM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX-resistant. 4. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides.

Cloning and functional characterisation of the mouse P2X7 receptor.

We have isolated a 1785-bp complementary DNA (cDNA) encoding the murine P2X7 receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80%, and 85% homology to the human and rat P2X7 subunits, respectively. Functional properties of the heterologously expressed murine P2X7 homomeric receptor broadly resembled those of the P2X7 receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X7 receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.

Molecular cloning and characterization of the rat P2Y4 receptor.

Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full-length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y4 receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2-methylthio-ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y4 receptor but with this significant difference in agonist pharmacology.

Plasticity of agonist binding sites in hetero-oligomers of the unitary glutamate receptor subunit XenU1.

Two subunits from Xenopus, XenNR1G and the "short" subunit XenU1, have previously been coexpressed to form a unitary (NMDA/non-NMDA type) glutamate receptor. We now show that an antibody to XenNR1G or an antibody to XenU1 precipitates the binding sites of both XenNR1G and XenU1, with the recombinant subunits or with solubilised Xenopus brain membranes, i.e., the combination occurs in vivo. The expressed XenU1 subunits are in the cell membrane and oriented correctly. XenU1 binds not only kainate with high affinity (K(D) 1.2 nM at 25 degrees C), but also the glycine site antagonist 5,7-dichlorokynurenic acid (DCKA). DCKA, GTP, or GTPgammaS displaces competitively all of the bound [3H]kainate, but glycine has no effect. The results suggest that a common binding site for kainate, DCKA, and GTP can exist on XenU1. In the XenNR1G/XenU1 complex, the kainate affinity is lowered eightfold, whereas the DCKA affinity is considerably increased (K(D) 147 nM). Only 18% of the binding to the complex has the properties of the NMDA receptor glycine site, the rest being due to switching of the high-affinity kainate site of XenU1 (low-affinity DCKA) to a high-affinity DCKA (low-affinity kainate) conformation. Surprisingly, a mammalian NR2 subunit can also combine with XenU1, and this introduces similar reciprocal changes in the binding of kainate and DCKA. The combined evidence suggests a common basic mode of agonist site formation in different subunit types of the ionotropic glutamate receptors.

Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR.

Studies using selective agonists have suggested that the contractile effect of extracellular nucleotides, such as ATP and UTP, in blood vessels is mediated mainly by P2X1 receptors with a smaller contribution of P2Y receptors while the mitogenic effect is mediated by P2Y (P2Y1, P2Y2, P2Y4, and P2Y6) receptors with no effect of P2X1 receptors. This indicates a difference in P2 receptor expression between the contractile and the synthetic phenotype of the SMC. To measure the expression of mRNA for these receptors a competitive RT-PCR assay was developed that utilised synthetic RNA-competitors allowing determination of the number of mRNA copies for each receptor in the samples. In the synthetic phenotype the mitogenic P2Y1 and P2Y2 receptor transcripts were upregulated by 342- and 8-fold, respectively, while the contractile P2X1 receptor is totally downregulated and the P2Y4 and P2Y6 receptors were unchanged. This plasticity of the receptor expression may be important in the transition from the contractile to the synthetic SMC phenotype.

Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts.

OBJECTIVES: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFB respond to extracellular ATP by inducing expression of c-fos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFB. METHODS: Expression of P2Y1 and P2Y2 receptors and induction of c-fos were examined by Northern blot analysis. CAFB growth was assessed by measuring [3H]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. RESULTS: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y1 and P2Y2) are expressed in cultured CAFB. Extracellular ATP induced the expression of c-fos mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [3H]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. CONCLUSIONS: These data demonstrate that CAFB express both P2Y1 and P2Y2 receptor mRNA and that CAFB respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [3H]thymidine incorporation into DNA and on expression of c-fos mRNA are exerted via distinct P2Y receptor subtypes.

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents.

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP >> GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.