Search

The extracellular ATP-gated cation channel P2X(2) is known to show voltage-dependent gating in spite of the absence of a canonical voltage sensor domain. We previously observed that the hyperpolarization-evoked activation of P2X(2) at the steady state in the presence of ATP varied depending on [ATP]. With increasing [ATP], the conductance-voltage (G-V) relationship shifted to more depolarized potentials and the activation kinetics were accelerated. Using a three-state model consisting of an ATP binding step and a rate limiting gating step, we successfully reproduced the voltage-dependent gating including its [ATP] dependence. In this study, in order to identify the structural basis of voltage and ATP dependence, we analysed various mutants in the Xenopus oocyte expression system under two-electrode voltage clamp. In the ATP binding region mutant K308R, the G-V relationship was shifted towards more hyperpolarized potentials, there was no clear [ATP] dependence, and activation was faster than in wild-type (WT). These results could be simulated by assuming an increase in the off rate of the gating step, in addition to changes in the ATP binding step. With F44C mutation in the 1st transmembrane (TM) region (TM1) or T339S in TM2, activation in low [ATP] was slow and the channel was constitutively active at all membrane potentials in high [ATP]. These results could be simulated by reducing the off rate of the gating step. Phenotypes of the double mutants, K308R/F44C and K308R/T339S, were similar to WT, suggesting that TM and ATP binding region mutants offset the effect of each other. Analysis of the effects on WT of two other agonists, ADP and AP(4)A, revealed that the electrostatic charge is not the sole critical factor. Taking these results together with the recently reported crystal structure, it was suggested that upon binding of ATP, the occupied binding site indirectly interacts with the extracellular end of the TM regions to trigger conformational changes for gating in a voltage-dependent manner.

The P2X(2) channel is a ligand-gated channel activated by ATP. Functional features that reflect the dynamic flexibility of the channel include time-dependent pore dilatation following ATP application and direct inhibitory interaction with activated nicotinic acetylcholine receptors on the membrane. We have been studying the mechanisms by which P2X(2) channel functionality is dynamically regulated. Using a Xenopus oocyte expression system, we observed that the pore properties, including ion selectivity and rectification, depend on the open channel density on the membrane. Pore dilatation was apparent when the open channel density was high and inward rectification was modest. We also observed that P2X(2) channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X(2) channels were activated upon membrane hyperpolarization. This voltage-dependent activation was also [ATP] dependent. With increases in [ATP], the speed of hyperpolarization-induced activation was increased and the conductance-voltage relationship was shifted towards depolarized potentials. Based on analyses of experimental data and various simulations, we propose that these phenomena can be explained by assuming a fast ATP binding step and a rate-limiting voltage-dependent gating step. Complete elucidation of these regulatory mechanisms awaits dynamic imaging of functioning P2X(2) channels.

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

Phosphoinositides (PIP(n)s) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X(2) channels also are regulated by PIP(n)s, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X(2) channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-d-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X(2) cytoplasmic domain to anionic lipids using PIP(n)s-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP(2)s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X(2) with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIP(n)s play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X(2) cytoplasmic C-terminal domain.

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X2 receptor subunit (P2X2-/-) and double mutant mice lacking both P2X2 and P2X3 subunits (P2X2/P2X3(Dbl-/-)), and compare these with previously characterized P2X3-/- mice. In patch-clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild-type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X2-/- mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X2/P2X3(Dbl-/-) mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X2 and P2X3 subunits. P2X2-/- and P2X2/P2X3(Dbl-/-) mice had reduced pain-related behaviours in response to intraplantar injection of formalin. Significantly, P2X3-/-, P2X2-/-, and P2X2/P2X3(Dbl-/-) mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X2-/- mice. Taken together, these data extend our findings for P2X3-/- mice, and reveal an important contribution of heteromeric P2X2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder.

Ligand-gated ion channels underlie and play important roles in synaptic transmission, and it is generally accepted that the ion channel pores have a rigid structure that enables strict regulation of ion permeation. One exception is the P2X ATP-gated channel. After application of ATP, the ion selectivity of the P2X2 channel time-dependently changes, i.e. permeability to large cations gradually increases, and there is significant cell-to-cell variation in the intensity of inward rectification. Here we show P2X2 channel properties are correlated with the expression level: increasing P2X2 expression level in oocytes increases permeability to large cations, decreases inward rectification and increases ligand sensitivity. We also observed that the inward rectification changed in a dose-dependent manner, i.e. when low concentration of ATP was applied to an oocyte with a high expression level, the intensity of inward rectification of the evoked current was weak. Taken together, these results show that the pore properties of P2X2 channel are not static but change dynamically depending on the open channel density. Furthermore, we identified by mutagenesis study that Ile328 located at the outer mouth of the pore is critical for the density-dependent changes of P2X2. Our findings suggest synaptic transmission can be modulated by the local density-dependent changes of channel properties caused, for example, by the presence of clustering molecules.

Vagal mechanoreceptors to the guinea-pig oesophagus, recorded extracellularly, in vitro, fired spontaneously at 3.3 +/- 0.2 Hz, (n = 75, from 57 animals), and had low thresholds to circumferential stretch. In this study, we have investigated whether mechanotransduction by intraganglionic laminar endings (IGLEs) directly relies on mechano-gated ion channels, or whether it is due to chemical activation by neurotransmitters (glutamate or ATP) released from other cells during mechanical distortion. Rapid distortion of focal transduction sites (IGLEs) evoked action potentials with a latency of < 10 ms. Antagonists to ionotropic (AP5, memantine and 6,7-dinitroquinoxaline-2,3-dione (DNQX)) and metabotropic glutamate receptors (N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) and (RS)-a-methyl-4-phosphono-phenylglycine (MPPG)) did not affect mechano-transduction. Glutamate, NMDA and the selective mGluR group II and III agonists, (2R, 4R)-APDC and L-AP4, had no effect on spontaneous or stretch-induced firing. The P2X purinoreceptor agonist, alpha,beta-methylene ATP, caused concentration-dependent excitation of vagal mechanoreceptors (EC50 = 22.2 microM) which was blocked by the non-selective P2 antagonist PPADS (30 microM). On its own, PPADS affected neither stretch-induced firing nor spontaneous firing. Neither Ca(2+)-free solution (1 mM EDTA, 3.6 mM Mg(2+)) solution nor Cd(2+) (100 microM) blocked stretch-induced firing. Thus chemical transmission is not involved in activation of vagal mechanoreceptors. The blocker of stretch-activated channels, Gd(3+) (300 microM), did not inhibit stretch-induced firing. However, benzamil (100 microM) significantly inhibited spontaneous and distension-evoked firing in a stretch-dependent manner; proportionally greater inhibition was seen with larger stretches. The results suggest that IGLEs of vagal tension receptors directly transduce mechanical stimuli probably via benzamil-sensitive, Gd3+-insensitive, stretch-activated ion channels, and that chemical transmission is not involved in transduction.

P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. ATP acts at P2X receptors to contribute to fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons but the subunit composition of enteric P2X receptors is unknown. These studies used tissues from P2X2 wild-type (P2X2+/+) and P2X2 gene knockout (P2X2-/-) mice to investigate the role of this subunit in enteric neurotransmission. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from ileal myenteric neurons in vitro. Drug-induced longitudinal muscle contractions and peristaltic contractions of ileal segments were also studied in vitro. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. RT-PCR analysis of mRNA from intestinal tissues and data from immunohistochemical studies verified P2X2 gene deletion. The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were reduced by mecamylamine (nicotinic cholinergic receptor antagonist) and PPADS (P2X receptor antagonist). The fEPSPs recorded from S neurons from P2X2-/- mice were unaffected by PPADS but were blocked by mecamylamine. ATP depolarized S and AH neurons from P2X2+/+ mice. ATP depolarized AH but not S neurons from P2X2-/- mice. alpha,beta-Methylene ATP (alpha,beta-mATP)(an agonist at P2X3 subunit-containing receptors) did not depolarize S neurons but it did depolarize AH neurons in P2X2+/+ and P2X2-/- mice. Peristalsis was inhibited in ileal segments from P2X2-/- mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X2+/+ and P2X2-/- mice. Gastrointestinal transit was similar in P2X2+/+ and P2X2-/- mice. It is concluded that P2X2 homomeric receptors contribute to fEPSPs in neural pathways underlying peristalsis studied in vitro.

We have studied the modulatory effect of dehydroepiandrosterone (DHEA), the most abundant neurosteroid produced by glial cells and neurones, on membrane currents induced by the activation of ionotropic ATP (P2X) receptors in neonatal rat dorsal root ganglion neurones. ATP (1 microM) induced three types of currents/responses termed F (fast and transient), S (slowly desensitizing) and M (mixed, sum of F- and S-type responses). DHEA (10 nM to 100 microM) concentration-dependently increased the amplitude of plateau-like currents of S- and M-type responses evoked by submaximal (1 microM) but not saturating (100 microM or 1 mM) concentrations of ATP. Alphabeta-methylene ATP (alphabetame-ATP, 5 microM) also evoked F-, S- and M-type responses, the plateau phases of which were potentiated by lowering external pH (6.3) and by ivermectin (IVM, 3 microM), indicating the presence heteromeric P2X2-containing receptors and possibly of functional native P2X4/6 receptors. There was a strict correlation between the potentiating effects of low pH and DHEA on alphabetame-ATP responses but not between that of IVM and DHEA, suggesting that DHEA selectively modulated P2X2-containing receptors. DHEA also potentiated putative homomeric P2X2 receptor responses recorded in the continuous presence of 1 microM 2'-(or 3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Our results constitute the first demonstration of a fast potentiation of P2X receptors by a neurosteroid and suggest that DHEA could be an endogenous modulator of P2X2-containing receptors thereby contributing to the facilitation of the detection and/or the transmission of nociceptive messages, particularly under conditions of inflammatory pain where the P2X receptor signalling pathway appears to be upregulated.

ATP is involved in central respiratory control and may mediate changes in the activity of medullary respiratory neurones during hypercapnia, thus playing an important role in central chemoreception. The main objective of this study was to explore further the role of ATP-mediated signalling in respiratory control and central chemoreception by characterising the profile of the P2X receptors expressed by physiologically identified respiratory neurones. In particular we determined whether respiratory neurones in the rostral ventrolateral medulla (VLM) express P2X2 receptor subunits of the ATP-gated ion channel, since ATP currents evoked at recombinant P2X2 receptors are potentiated by lowering extracellular pH. Experiments were performed on anaesthetised (pentobarbitone sodium 60 mg kg-1 I.P., then 10 mg kg-1 I.V. as required), gallamine-triethiodide-treated (10 mg kg-1 I.V., then 2-4 mg kg-1 h-1 I.V.) and artificially ventilated rats. The VLM respiratory neurones were classified according to the timing of their discharge pattern in relation to that of the phrenic nerve and by the exclusion of pump cells from the study population; these were labelled with Neurobiotin using the juxtacellular method, and visualised with fluorescence microscopy. It was found that a substantial proportion of the VLM respiratory neurones express the P2X2 receptor subunit. P2X2 receptor subunit immunoreactivity was detected in approximately 50 % (six out of 12) of expiratory neurones and in approximately 20 % (two out of 11) of neurones with inspiratory-related discharge (pre-inspiratory and inspiratory). In contrast, no Neurobiotin-labelled VLM respiratory neurones (n = 19) were detectably immunoreactive for the P2X1 receptor subunit. Microionophoretic application of ATP (0.2 M, 20-80 nA for 40 s) increased the activity of approximately 80 % (13 out of 16) of expiratory neurones and of approximately 30 % (five out of 18) of VLM neurones with inspiratory-related discharge. These effects were abolished by the P2 receptor blocker suramin (0.02 M, 80 nA), which also reduced the baseline firing in some expiratory neurones. These data indicate that modulation of P2X2 receptor function, such as that evoked by acidification of the extracellular environment during hypercapnia, may contribute to the changes in activity of the VLM respiratory neurones that express these receptors.

P2X(2) receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X(2) receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC(50) of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 microM ATP of wild-type P2X(2) and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the pK(a) (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X(2) and revealed a substantial pH-dependent decrease in the maximum response with a pK(a) near 6.0. Substituting a lysine for H319 reduced the EC(50) for ATP 40-fold. Zinc (20 microM) potentiated the responses to 10 microM ATP of wild-type P2X(2) and seven histidine mutants by approximately 8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.

1. Hypoxic chemotransmission in the rat carotid body (CB) is mediated in part by ATP acting on suramin-sensitive P2X purinoceptors. Here, we use RT-PCR, cloning and sequencing techniques to show P2X2 and P2X3 receptor expression in petrosal neurones, some of which develop functional chemosensory units with CB receptor clusters in co-culture. 2. Single-cell RT-PCR revealed that hypoxia-responsive neurones, identified electrophysiologically in co-culture, expressed both P2X2 and P2X3 mRNA. 3. Isohydric hypercapnia (10 % CO(2); pH 7.4) caused excitation of chemosensory units in co-culture. This excitation depended on chemical transmission, with ATP acting as a co-transmitter, since it was inhibited by reduction of the extracellular Ca(2+):Mg(2+) ratio and by the purinoceptor blocker suramin (50-100 microM). 4. Hypoxia and isohydric hypercapnia could separately excite the same chemosensory unit, and together the two stimuli interacted synergistically. 5. Using confocal immunofluorescence, co-localization of P2X2 and P2X3 protein was demonstrated in many petrosal somas and CB afferent terminals in situ. Taken together, these data indicate that ATP and P2X2-P2X3 purinoceptors play important roles in the peripheral control of respiration by carotid body chemoreceptors.

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

1. The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP-evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole-cell patch clamp technique. 2. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na(+) > Li(+) > Cs(+) > Rb(+) > K(+), and permeability ratios relative to Cs(+) (P(X)/P(Cs)) ranging from 1.11 to 0.86. 3. The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca(2+) > Sr(2+) > Ba(2+) > Mn(2+) > Mg(2+). ATP-evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased. 4. The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size. 5. Acidification to pH 6.2 increased the ATP-induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti-P2X(2) and/or anti-P2X(4) but not anti-P2X(1) antibodies attenuated the ATP-evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X(2) and P2X(4) receptor subtypes expressed in rat submandibular neurones. 6. The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X(2) receptors expressed in mammalian cells.

1. Characterization of P2X receptors on neurons of guinea-pig superior cervical ganglion (SCG) has been carried out using a whole-cell voltage-clamp technique. 2. Application of ATP and alpha,beta-methylene ATP (alphabeta-MeATP) produced fast activating inward currents, which desensitized slowly. The maximum response to alphabeta-MeATP was 36 +/- 23 % (range 0.1-100 %) of that evoked by ATP in the same cell. 3. Co-application of alphabeta-MeATP (300 microM) with ATP (300 microM) produced a response that was 97 +/- 1 % of that given by ATP alone. Following desensitization with alphabeta-MeATP, the decrease in response to ATP was equal to the absolute reduction in response to alphabeta-MeATP in the same cell. 4. The concentration-response curve for alphabeta-MeATP had an EC50 of 42 microM and a Hill coefficient of 1.17. For cells where the ratio of alphabeta-MeATP/ATP currents at 100 microM was < 0.1, the ATP concentration-response curve had an EC50 of 56 microM and a Hill coefficient of 1.95. However, in cells where the ratio was > 0.7, the curve had an EC50 of 60 microM and a Hill coefficient of 0.97. 5. The response to 100 microM alphabeta-MeATP was inhibited by 2' (or 3')-O-trinitrophenyl-ATP (TNP-ATP) with an IC50 of 70 nM. However, on cells where the ratio of alphabeta-MeATP/ATP currents was < 0.1, ATP was inhibited by TNP-ATP with an IC50 of 522 nM. 6. Immunohistochemical staining with antibodies raised against rat P2X2 and P2X3 epitopes suggested that both subunits were expressed by guinea-pig SCG neurons. 7. We conclude that varying proportions of two distinct P2X receptors coexist on the cell bodies of individual guinea-pig SCG neurons, which may correspond to homomeric P2X2 and heteromeric P2X2/3 receptors.

1. P2X2 channels are activated by extracellular ATP. Despite being commonly described as non-desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2. The ability to inactivate whole-cell currents developed slowly after breaking in. In contrast, currents from excised patches exhibited rapid (approximately 100 ms) inactivation with a dependence on extracellular Ca2+, ATP and voltage. 3. The inactivation rate increased with the fourth power of [Ca2+] suggesting that the functional channel may be a tetramer. Ca2+ had both a higher affinity and a larger Hill coefficient for inactivation than Mg2+, Ba2+ or Mn2+. Trivalent cations at concentrations up to the solubility product of ATP had no effect. The change in apparent co-operativity with ionic species suggests the presence of experimentally unresolved ligand-insensitive kinetic steps. 4. Based on the weak voltage dependence of inactivation and the lack of effect of intracellular Ca2+ buffers, the Ca2+-binding sites are probably located near the extracellular surface of the membrane. 5. The recovery from inactivation was slow, with a time constant of approximately 7 min. 6. Ca2+-sensitive inactivation only appeared when the membrane was disrupted in some manner. Treatment with actin and microtubule reagents did not induce inactivation, suggesting that an intact cytoskeleton is not necessary. 7. Inactivation rates observed in different patch configurations suggest that the induction of Ca2+-dependent inactivation was due to the loss of a diffusible cofactor located in the membrane or the cytoplasm.

1. The ATP-activated P2X2(a) and P2X2(b) receptor splice variants, which differ only in their C-terminal sequences, desensitize at different rates. We used mutational analysis to investigate the involvement of the C-terminal region in receptor desensitization. Rat wild-type and mutant P2X2 receptors were expressed in Xenopus oocytes and currents were measured using the two-electrode voltage-clamp technique. 2. Truncating P2X2 at the Lys369 splice site increased the rate of desensitization by >100-fold. Recovery from desensitization was slowed by approximately 5-fold. 3. Addition of Val370 onto the C-terminus of the truncated receptor slowed desensitization by approximately 70-fold. Point mutations that substituted either smaller or larger hydrophobic amino acids for Val370, within the P2X2(a) splice variant, had profound effects on the rate of desensitization. The rate decreased with increasing hydrophobicity but was not dependent upon the precise structure of the side group. 4. A mutant receptor, with only nine amino acids, Val-Asp-Pro-Lys-Gly-Leu-Ala-Gln-Leu, beyond the Lys369 splice site, desensitized at a similar rate to P2X2(a). Injection of the peptide of this sequence into oocytes expressing P2X2(a) increased the rate of desensitization, whereas the eight-residue peptide lacking the valine had no effect. 5. Neutralizing lysines in the vicinity of the splice site increased the rate of receptor desensitization. Substituting glutamine for Lys365 produced the greatest effect ( approximately 30-fold increase), whereas mutating lysines that were further upstream or downstream of this position had progressively less of an effect. 6. We conclude that the C-terminal splice site of the P2X2 receptor is located within a region that is critically involved in regulating the rate of receptor desensitization. The valine at position 370 interacts with an intracellular hydrophobic site to slow the rate of desensitization. Nearby lysines may facilitate this interaction.

1. The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura-2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2. ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1-100 microM range (EC50 = 4.8 microM). This effect was observed in only approximately 40 % of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 microM), evoked AVP, but no significant OT, release from these terminals. 4. Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP-induced responses. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP-induced [Ca2+]i response. 5. To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2-methylthio-ATP > ATP-gamma-S > alpha, beta-methylene-ATP > ADP. The compounds adenosine, AMP, beta, gamma-methylene-ATP and UTP were ineffective. 6. The ATP-dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence-quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP-induced [Ca2+]i increase and AVP release were independent of and additive to a K+-induced response, in addition to being insensitive to Cd2+. The ATP-induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage-dependent Ca2+ channel. 7. We propose that ATP, co-released with neuropeptides, could act as a paracrine-autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.

1. The ATP receptor subunit P2X2 was expressed in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. ATP-activated currents were studied with two-electrode voltage clamp recordings from oocytes, whole-cell recordings from HEK 293 cells, and outside-out patch clamp recordings from both cell types. The steady-state current-voltage (I-V) relation showed profound inward rectification in all recording configurations. 2. Recordings from outside-out patches demonstrated that inward rectification does not require intracellular Mg2+ or polyamines, and that inward rectification was present when the same solution was used on both sides of the patch. 3. Voltage jump experiments were performed to evaluate the voltage dependence of channel gating. After fast voltage jumps, instantaneous current jumps were followed by substantial relaxations to the steady state. The time course of the current relaxations could be fitted by single exponential functions. The instantaneous I-V relation was less inwardly rectifying than the steady-state I-V relation; however, it was not linear. 4. Single channel recordings indicated that the single channel conductance became smaller when the membrane potential became more positive. This decrease could quantitatively account for inward rectification of the instantaneous I-V relation. 5. We conclude that inward rectification of P2X2 is due to two mechanisms: voltage-dependent gating and voltage dependence of the single channel conductance.