A P2X receptor-mediated nociceptive afferent pathway to lamina I of the spinal cord

Of the six lamina regions in the dorsal horn of the spinal cord, lamina I is a major sensory region involved in nociceptive transmission under both physiological and pathological conditions. While P2X receptors have been shown to be involved in nociception, it remains unknown if P2X receptors are involved in nociceptive transmission to lamina I neurons. Using rat spinal cord slice preparations and patch-clamp recordings, we have demonstrated that the excitatory synaptic transmission between primary afferent fibers and lamina I neurons is significantly affected by ATP and α,β-methylene-ATP. The synaptic effects of them include the increases of the frequency of both miniature excitatory postsynaptic currents (mEPSCs) and spontaneous EPSCs (sEPSCs), and decreases of evoked EPSCs (eEPSCs). These effects were blocked by pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS, 10 μM) and suramin (30 μM). In the neurons for which ATP and α,β-methylene-ATP had effects on mEPSCs, sEPSCs and eEPSCs, capsaicin produced similar synaptic effects. Our results indicate that P2X receptors are expressed on many afferent fibers that directly synapse to lamina I neurons. Furthermore, these P2X receptor-expressing afferent fibers are capsaicin-sensitive nociceptive afferents. Thus, this study reveals a P2X receptor-mediated nociceptive afferent pathway to lamina I of the spinal cord and provides a new insight into the nociceptive functions of P2X receptors

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking

Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

The ATP-gated P2X4 receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X4 receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X4 receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X4 receptors and a functional analysis revealed that P2X4 receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X4 receptors revealed two different forms of activated structures under 0 Ca2+ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X4 receptors display pore dilation in the absence of Ca2+. With Ca2+, the P2X4 receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X4 receptors that correlate with pore dynamics

Effects of thermal fluctuation and the receptor-receptor interaction in bacterial chemotactic signalling and adaptation

Bacterial chemotaxis is controlled by the conformational changes of the receptors, in response to the change of the ambient chemical concentration. In a statistical mechanical approach, the signalling due to the conformational changes is a thermodynamic average quantity, dependent on the temperature and the total energy of the system, including both ligand-receptor interaction and receptor-receptor interaction. This physical theory suggests to biology a new understanding of cooperation in ligand binding and receptor signalling problems. How much experimental support of this approach can be obtained from the currently available data? What are the parameter values? What is the practical information for experiments? Here we make comparisons between the theory and recent experimental results. Although currently comparisons can only be semi-quantitative or qualitative, consistency is clearly shown. The theory also helps to sort a variety of data.Comment: 26 pages, revtex. Journal version. Analysis on another set of data on adaptation time is adde

The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors. © 2013 Sun et al

Excitatory effect of ATP on rat area postrema neurons

ATP-induced inward currents and increases in the cytosolic Ca2+ concentration ([Ca]in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (IATP) and [Ca]in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the IATP exhibited a strong inward rectification, and the amplitude of the IATP was concentration-dependent. The IATP was markedly reduced in the absence of external Na+, and the addition of Ca2+ to Na+-free saline increased the IATP. ATP did not increase [Ca]in in the absence of external Ca2+, and Ca2+ channel antagonists partially inhibited the ATP-induced [Ca]in increase, indicating that ATP increases [Ca]in by Ca2+ influx through both P2XR channels and voltage-dependent Ca2+ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at Vhs between −70 and −10 mV when the IATP and ACh-induced current (IACh) were inward, but no occlusion was observed when these currents were outward at a Vh of +40 mV. The IATP was not inhibited by co-application of ACh when the IACh was markedly decreased either by removal of permeant cations, by setting Vh close to the equilibrium potential of IACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels

Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201