P2X3 receptors and peripheral pain mechanisms

ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X₃ subunits are found in a subset of small-diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo-oligomeric channels, or they can assemble with P2X₂ subunits into hetero-oligomers. Studies with antagonists selective for P2X₃-containing receptors, experiments with antisense oligonucleotides to reduce P2X₃ subunit levels, and behavioural testing of P2X₃ knock-out mice, all suggest a role for the P2X_2/3 receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones.     

Density-dependent changes of the pore properties of the P2X2 receptor channel

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.     

Dynamic aspects of functional regulation of the ATP receptor channel P2X2

The P2X₂ 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 P2X₂ 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 P2X₂ channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X₂ 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 P2X₂ channels.     

Dehydroepiandrosterone Potentiates Native Ionotropic ATP Receptors Containing the P2X2 Subunit in Rat Sensory Neurones

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 μ m ) 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 n m to 100 μ m ) concentration-dependently increased the amplitude of plateau-like currents of S- and M-type responses evoked by submaximal (1 μ m ) but not saturating (100 μ m or 1 mM) concentrations of ATP. αβ-Methylene ATP (αβme-ATP, 5 μ m ) 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 μ m ), indicating the presence heteromeric P2X₂-containing receptors and possibly of functional native P2X_4/6 receptors. There was a strict correlation between the potentiating effects of low pH and DHEA on αβme-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 μ m 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.     

The role of histidine residues in modulation of the rat P2X2 purinoceptor by zinc and pH

P2X₂ 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 P2X₂ 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 EC₅₀ of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 μ m ATP of wild-type P2X₂ 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 p K _a (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X₂ and revealed a substantial pH-dependent decrease in the maximum response with a p K _a near 6.0. Substituting a lysine for H319 reduced the EC₅₀ for ATP 40-fold. Zinc (20 μ m ) potentiated the responses to 10 μ m ATP of wild-type P2X₂ and seven histidine mutants by ∼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.     

Regulation of histamine release from human basophil leucocytes: role of H1, H2 and H3 receptors

A novel class of histamine receptors (H3), controlling histamine synthesis and release, was described in rat and human brain and peripheral nerve endings. The present study was undertaken to evaluate whether H3 receptors contribute to the regulation of histamine release from human basophils. Basophil leucocytes were incubated with a H3 antagonist (thioperamide; concentrations ranging from 1 nM to 10 microM) or with a H3 ((R)alpha methyl-histamine; concentrations ranging from 1 to 100 mM), and subsequently were stimulated with optimal doses of anti-IgE and formyl-methionyl-leucyl-phenyl-alanine (f-met peptide). No significant modifications of histamine release were observed after incubation either with the H3 agonist or with the H3 antagonist. By contrast, a H2 antagonist (cimetidine; concentrations ranging from 1 to 100 microM) exerted a dose-dependent enhancing effect on anti-IgE- and, to a lesser extent, on f-met peptide-induced histamine release. A H1 antihistamine (chlorpheniramine; concentrations ranging from 100 nM to 1 microM), at the highest concentration employed, displayed an inhibitory activity on IgE-dependent and IgE-independent histamine release. Exogenous histamine was shown to exert a dose-dependent inhibitory effect on two-staged anti-IgE-induced histamine release. Taken as a whole, these results suggest that H3 receptors are not involved in the regulation of histamine release from human basophils; by contrast, H2 receptors participate in controlling histamine release from human basophils, as previously demonstrated by other authors.