Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors

Two histidines are known to be essential for zinc potentiation of rat P2X₂ receptors, but the chemistry of zinc coordination would suggest that other residues also participate in this zinc-binding site. There is also a second lower affinity zinc-binding site in P2X₂ receptors whose constituents are unknown. To assess whether the extracellular acidic residues of the P2X₂ receptor contribute to zinc potentiation or inhibition, site-directed mutagenesis was used to produce alanine substitutions at each extracellular glutamate or aspartate. Two electrode voltage clamp recordings from Xenopus oocytes indicated that 7 of the 34 mutants (D82A, E85A, E91A, E115A, D136A, D209A, and D281A) were deficient in zinc potentiation and one mutant (E84A) was deficient in zinc inhibition. Additional tests on cysteine mutants at these eight positions indicated that D136 is the only residue that is a strong candidate to be at the potentiating zinc-binding site, and that E84 is unlikely to be at the inhibitory zinc-binding site.     

The role of positively charged amino acids in ATP recognition by human P2X(1) receptors

P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.     

Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP

The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.     

Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X(1) receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X(2) and P2X(4) receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X(2) K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.     

Contribution of conserved glycine residues to ATP action at human P2X1 receptors: mutagenesis indicates that the glycine at position 250 is important for channel function

Glycine residues can introduce flexibility in proteins, give rise to turns and breaks in secondary structure and are key components of some nucleotide binding motifs. In the P2X receptor extracellular ATP binding domain, 11 glycine residues are completely conserved and an additional five are conserved in at least five of the seven family members. We have mutated individual conserved glycine residues and determined their effect on the ATP sensitivity and time-course of P2X1 receptors expressed in Xenopus oocytes. In the majority of cases, replacement by alanine had no or a less than 3-fold effect on ATP sensitivity and time-course of responses. G71A resulted in a 6-fold decrease in ATP potency and ATP (10 mM) failed to evoke functional responses from G96A, G250A and G301A mutant receptors. However, proline or cysteine could substitute for glycine at positions 96 and 301, giving receptors that were essentially normal. At glycine 250 substitution by serine gave functional responses to ATP with no effect on ATP sensitivity but a reduction in peak amplitude; in contrast, functional responses were not recorded when glycine 250 was replaced by the amino acids alanine, cysteine, aspartate, phenylalanine, isoleucine, lysine, proline or asparagine. These results suggest that glycine 250 plays an important role in determining the function of P2X receptors.     

Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors).

Extracellular adenosine 5'-triphosphate (ATP) has been reported to produce excitatory actions in the nervous system, such as excitatory postsynaptic potentials or currents in both central and peripheral neurons, via activation of a class of ATP-gated membrane ion channels designated P2X receptors. This article reviews studies of alcohol effects on these receptor-channels. Ethanol has been found to inhibit ATP-gated ion channel function by shifting the agonist concentration-response curve to the right in a parallel manner, increasing the EC50 without affecting Emax of this curve. To distinguish whether this inhibition involves competitive antagonism of agonist action or a decrease in the affinity of the agonist binding site, the kinetics of activation and deactivation of agonist-activated current were studied. Ethanol was found to decrease the time-constant of deactivation of ATP-gated ion channels without affecting the time-constant of activation, indicating that ethanol inhibits the function of these receptors by an allosteric decrease in the affinity of the agonist binding site. The inhibition of ATP-gated ion channel function by a number of alcohols was found to exhibit a distinct cutoff effect that appeared to be related to the molecular volume of the alcohols. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. This cutoff effect has been interpreted as evidence that alcohols inhibit the function of ATP-gated ion channels by interacting with a hydrophobic pocket of circumscribed dimensions on the receptor protein. To evaluate the localization of this presumed alcohol binding site, the effect of the intracellular application of ethanol was studied on the inhibition of ATP-activated current by extracellularly applied ethanol. The intracellular application of 100 mM ethanol did not affect the inhibition of current by 100 mM extracellular ethanol, suggesting that the alcohol inhibition of ATP-gated ion channel function involves the extracellular domain of the receptor. Finally, recent studies suggest that the alcohol sensitivity of ATP-gated channels may be regulated by physiological mechanisms.     

Molecular physiology of P2X receptors and ATP signalling at synapses

ATP is found in every cell, where it is a major source of energy. But in the nervous system, ATP also has additional actions, which include its role in fast synaptic transmission and modulation. Here I discuss the 'fast' actions of ATP at synapses, the properties of the receptors that are activated by ATP and the physiology of ATP signalling, with emphasis on its role in pain processing.     

Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.     

Role of aromatic and charged ectodomain residues in the P2X(4) receptor functions

The localization of ATP binding site(s) at P2X receptors and the molecular rearrangements associated with opening and closing of channels are still not well understood. At P2X(4) receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated low or non-responsive mutants, whereas the F294A mutant was functional. The loss of receptor function was also observed in K67R, R295K, and K313R mutants, but not in F185W, K190R, F230W, R278K, and D280E mutants. To examine whether the loss of function reflects decreased sensitivity of mutants for ATP, we treated cells with ivermectin, an antiparasitic agent that enhances responsiveness of P2X(4)R. In the presence of ivermectin, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with the EC(50) values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 micromol/L at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A and K67R mutants, respectively. These results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three-dimensional structure of P2X(4)R for agonist binding and/or channel gating.     

The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (DeltaPsi) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, DeltaPsi generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, DeltaPsi generation and consequent large-scale ROS production.     

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.     

env-encoded residues are not required for transformation by p48v-myb.

The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus.     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize

P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X(2) receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm(3)) also points to a trimeric structure. P2X(2) receptors bearing His(6) epitope tags were incubated with anti-His(6) antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123 degrees , again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X(6) receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm(3). We conclude that P2X(2) receptors are trimers, whereas the P2X(6) receptor subunits do not form stable oligomers.     

P2X1 receptors and the endothelium

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, alpha, beta methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.     

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function

Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.