Synthesis and in vitro evaluation of novel compounds and discovery of a promising iodine-125 radioligand for purinergic P2X7 receptor (P2X7R).

The purinergic P2X ligand-gated ion channel 7 receptor (P2X7R) plays a critical role in various inflammatory processes and other diseases. Fast determination of compounds P2X7R binding potency and discovery of a promise PET radiotracer for imaging P2X7R require a P2X7R suitable radioligand for radioactive competitive binding assay. Herein, we designed and synthesized thirteen new P2X7R ligands and determined the in vitro binding potency.

Modulation of Microglial Function by ATP-Gated P2X7 Receptors: Studies in Rat, Mice and Human.

P2X receptors are a family of seven ATP-gated ion channels that trigger physiological and pathophysiological responses in a variety of cells. Five of the family members are sensitive to low concentrations of extracellular ATP, while the P2X6 receptor has an unknown affinity. The last subtype, the P2X7 receptor, is unique in requiring millimolar concentrations to fully activate in humans.

Using Whole-Cell Electrophysiology and Patch-Clamp Photometry to Characterize P2X7 Receptor Currents.

The fundamental property of P2X7 receptor (P2X7R) channels is the transport of cations across the cell surface membrane. Electrophysiology and patch-clamp photometry are readily accessible methods of measuring this flux in a wide range of cell types. They are important tools used to characterize the functional properties of native cells studied in cell culture, in vitro tissue slices, and, in some cases, in situ single cells.

Physiology of Cultured Human Microglia Maintained in a Defined Culture Medium.

Microglia are the primary immune cell of the CNS, comprising 5-20% of the ∼60 billion neuroglia in the human brain. In the developing and adult CNS, they preferentially target active neurons to guide synapse maturation and remodeling. At the same time, they are the first line of defense against bacterial, fungal, and viral CNS infections.

Ca flux through splice variants of the ATP-gated ionotropic receptor P2X7 is regulated by its cytoplasmic N terminus.

Activation of ionotropic P2X receptors increases free intracellular Ca ([Ca] ) by initiating a transmembrane cation flux. We studied the "a" and "k" splice variants of the rat purinergic P2X7 receptor (rP2X7aR and rP2X7kR) to exhibit a significant difference in Ca flux through this channel. This difference is surprising because the variants share absolute sequence identity in the area of the pore that defines ionic selectivity.

ATP-Gated P2X7 Receptors Require Chloride Channels To Promote Inflammation in Human Macrophages.

Immune cells of myeloid origin show robust expression of ATP-gated P2X7 receptors, two-transmembrane ion channels permeable to Na, K, and Ca Receptor activation promotes inflammasome activation and release of the proinflammatory cytokines IL-1β and IL-18. In this study, we show that ATP generates facilitating cationic currents in monocyte-derived human macrophages and permeabilizes the plasma membrane to polyatomic cationic dyes. We find that antagonists of PLA and Cl channels abolish P2X7 receptor-mediated current facilitation, membrane permeabilization, blebbing, phospholipid scrambling, inflammasome activation, and IL-1β release.

A central role for P2X7 receptors in human microglia.

Background: The ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1β release. However, the role of the P2X7R in primary adult human microglia is poorly understood.

Synthesis and in vitro characterization of a P2X7 radioligand [I]TZ6019 and its response to neuroinflammation in a mouse model of Alzheimer disease.

The purinergic receptor P2X ligand-gated ion channel 7 (P2X7 receptor) is a promising imaging target to detect neuroinflammation. Herein, we report development of a potent iodinated radiotracer for P2X7 receptor, [I]TZ6019. The radiosynthesis of [I]TZ6019 was accomplished by allylic-tin precursor iodination using [I]NaI with good radiochemical yield of 85% and high radiochemical purity of > 99%.

P2X receptor overexpression induced by soluble oligomers of amyloid beta peptide potentiates synaptic failure and neuronal dyshomeostasis in cellular models of Alzheimer's disease.

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aβ). Because Aβ increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aβ and the ability of P2XRs to modulate network synaptic tone.

Pharmacologic characterizations of a P2X7 receptor-specific radioligand, [11C]GSK1482160 for neuroinflammatory response.

Objective: The P2X7 receptor (P2X7R) is a key regulatory element in the neuroinflammatory cascade that provides a promising target for imaging neuroinflammation. GSK1482160, a P2X7R modulator with nanomolar binding affinity and high selectivity, has been successfully radiolabeled and utilized for imaging P2X7 levels in a mouse model of lipopolysaccharide-induced systemic inflammation. In the current study, we further characterized its binding profile and determined whether [C]GSK1482160 can detect changes in P2X7R expression in a rodent model of multiple sclerosis.

CLIC1 null mice demonstrate a role for CLIC1 in macrophage superoxide production and tissue injury.

We generated and studied CLIC1 null (C1KO) mice to investigate the physiological role of this protein. C1KO and matched wild-type (WT) mice were studied in two models of acute toxic tissue injury. CLIC1 expression is upregulated following acute injury of WT kidney and pancreas and is absent in C1KOs.

HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)).

Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein.

Engagement of the GABA to KCC2 signaling pathway contributes to the analgesic effects of A3AR agonists in neuropathic pain.

More than 1.5 billion people worldwide suffer from chronic pain, yet current treatment strategies often lack efficacy or have deleterious side effects in patients. Adenosine is an inhibitory neuromodulator that was previously thought to mediate antinociception through the A1 and A2A receptor subtypes.

Quantifying Ca2+ current and permeability in ATP-gated P2X7 receptors.

ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca(2+) through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca(2+) is universally acknowledged, the biophysics of the Ca(2+) flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca(2+) permeability are difficult to apply to P2X7 receptors.

Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin.

P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments.

Principles and properties of ion flow in P2X receptors.

P2X receptors are a family of trimeric ion channels that are gated by extracellular adenosine 5'-triphosphate (ATP). These receptors have long been a subject of intense research interest by virtue of their vital role in mediating the rapid and direct effects of extracellular ATP on membrane potential and cytosolic Ca(2+) concentration, which in turn underpin the ability of ATP to regulate a diverse range of clinically significant physiological functions, including those associated with the cardiovascular, sensory, and immune systems. An important aspect of an ion channel's function is, of course, the means by which it transports ions across the biological membrane.

Allosteric modulation of Ca2+ flux in ligand-gated cation channel (P2X4) by actions on lateral portals.

Human P2X receptors are a family of seven ATP-gated ion channels that transport Na(+), K(+), and Ca(2+) across cell surface membranes. The P2X4 receptor is unique among family members in its sensitivity to the macrocyclic lactone, ivermectin, which allosterically modulates both ion conduction and channel gating. In this paper we show that removing the fixed negative charge of a single acidic amino acid (Glu(51)) in the lateral entrance to the transmembrane pore markedly attenuates the effect of ivermectin on Ca(2+) current and channel gating.

Calcium-dependent decrease in the single-channel conductance of TRPV1.

TRPV1 is a Ca(2+) permeable cation channel gated by multiple stimuli including noxious heat, capsaicin, protons, and extracellular cations. In this paper, we show that Ca(2+) causes a concentration and voltage-dependent decrease in the capsaicin-gated TRPV1 single-channel conductance. This Ca(2+)-dependent effect on conductance was strongest at membrane potentials between -60 and +20 mV, but was diminished at more hyperpolarised potentials.

Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).

P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain.

Pannexin 1 is the conduit for low oxygen tension-induced ATP release from human erythrocytes.

Erythrocytes release ATP in response to exposure to the physiological stimulus of lowered oxygen (O(2)) tension as well as pharmacological activation of the prostacyclin receptor (IPR). ATP release in response to these stimuli requires activation of adenylyl cyclase, accumulation of cAMP, and activation of protein kinase A. The mechanism by which ATP, a highly charged anion, exits the erythrocyte in response to lowered O(2) tension or receptor-mediated IPR activation by iloprost is unknown.

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.

The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta.

Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry.

Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca(2+), although the absolute magnitude of the Ca(2+) current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca(2+) to total current through native and recombinant ASIC1a receptors.

Tunable calcium current through TRPV1 receptor channels.

TRPV1 receptors are polymodal cation channels that open in response to diverse stimuli including noxious heat, capsaicin, and protons. Because Ca2+ is vital for TRPV1 signaling, we sought to precisely measure its contribution to TRPV1 responses and discovered that the Ca2+ current was tuned by the mode of activation. Using patch clamp photometry, we found that the fraction of the total current carried by Ca2+ (called the Pf%) was significantly smaller for TRPV1 currents evoked by protons than for those evoked by capsaicin.