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. This low sensitivity imparts the agonist with the ability to act as a damage-associated molecular pattern that triggers the innate immune response in response to the elevated levels of extracellular ATP that accompany inflammation and tissue damage. In this review, we focus on microglia because they are the primary immune cells of the central nervous system, and they activate in response to ATP or its synthetic analog, BzATP. We start by introducing purinergic receptors and then briefly consider the roles that microglia play in neurodevelopment and disease by referencing both original works and relevant reviews. Next, we move to the role of extracellular ATP and P2X receptors in initiating and/or modulating innate immunity in the central nervous system. While most of the data that we review involve work on mice and rats, we highlight human studies of P2X7R whenever possible.

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. Further, they are efficient methods of probing the relation of structure to function of recombinant receptors expressed in heterologous systems. Here, we provide step-by-step procedures for use of two standard recording protocols, broken-patch and perforated-patch voltage clamp. Further, we describe a third technique, called the dye-overload method, that uses simultaneous measurement of membrane current and fura-2 fluorescence to quantify the contribution of Ca2+ flux to the ATP-gated current.

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. Although an extensive literature details their roles in rodents, less is known about how they function in humans because of the difficulty in obtaining tissue samples and the understandable inability to extensively study human microglia in situ. In this study, we use recent advances in the study of brain microenvironments to establish cultures of primary human microglia in a serum-free medium. Postsurgical samples of human brain were enzymatically and mechanically dissociated into single cells, and microglia were isolated at high purity by positive selection using CD11b Ab-coated microbeads. The CD11b+ cells were plated on poly-l-lysine-coated surfaces and bathed in serum-free DMEM/F12 supplemented with three essential components (TGF-ß, IL-34, and cholesterol). Under these conditions, microglia assumed a ramified morphology, showed limited proliferation, actively surveyed their surroundings, and phagocytosed bacterial microparticles. In the presence of LPS, they assumed a more compact shape and began production of proinflammatory cytokines and reactive oxygen species. LPS on its own triggered release of TNF-α, whereas release of IL-1ß required costimulation by ATP. Thus, human microglia maintained in a defined medium replicate many of the characteristics expected of native cells in the brain and provide an accessible preparation for investigations of human microglial physiology, pharmacology, and pathophysiology.

Ca2+ 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 Ca2+ ([Ca2+] i ) 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 Ca2+ 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. Here, we used patch-clamp fluorometry and chimeric receptors to show that the fraction of the total current carried by Ca2+ is a function of the primary sequence of the cytoplasmic N terminus. Using scanning mutagenesis, we identified five sites within the N terminus that respond to mutagenesis with a decrease in fractional calcium current and an increase in permeability to the polyatomic cation, N-methyl-d-glucamine (NMDG+), relative to Na+ (PNMDG/PNa). We tested the hypothesis that these sites line the permeation pathway by measuring the ability of thiol-reactive MTSET+ to alter the current of cysteine-substituted variants, but we detected no effect. Finally, we studied the homologous sites of the rat P2X2 receptor (rP2X2R) and observed that substitutions at Glu17 significantly reduced the fractional calcium current. Taken together, our results suggest that a change in the structure of the N terminus alters the ability of an intra-pore Ca2+ selectivity filter to discriminate among permeating cations. These results are noteworthy for two reasons: they identify a previously unknown outcome of mutagenesis of the N-terminal domain, and they suggest caution when assigning structure to function for truncated P2X receptors that lack a part of the N terminus.

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 Ca2+ Receptor activation promotes inflammasome activation and release of the proinflammatory cytokines IL-1ß 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 PLA2 and Cl- channels abolish P2X7 receptor-mediated current facilitation, membrane permeabilization, blebbing, phospholipid scrambling, inflammasome activation, and IL-1ß release. Our data demonstrate significant differences in the actions of ATP in murine and human macrophages and suggest that PLA2 and Cl- channels mediate innate immunity downstream of P2X7 receptors in human macrophages.

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-1ß release. However, the role of the P2X7R in primary adult human microglia is poorly understood. METHODS: We used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels. RESULTS: We found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl- channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl- channel antagonists. CONCLUSIONS: Our results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.

Synthesis and in vitro characterization of a P2X7 radioligand [123I]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, [123I]TZ6019. The radiosynthesis of [123I]TZ6019 was accomplished by allylic-tin precursor iodination using [123I]NaI with good radiochemical yield of 85% and high radiochemical purity of > 99%. Human embryonic kidney 293 (HEK-293) cell line stably transfected with the human P2X7 receptor was used to characterize the binding affinity of TZ6019 by fluorescence, radioactive competitive, and saturation binding assays. A radioligand competitive binding assay with [123I]TZ6019 demonstrated that the nonradioactive compound TZ6019 has an IC50 value of 9.49 ± 1.4nM, and the known P2X7 receptor compound GSK1482160 has an IC50 value of 4.30 ± 0.86nM, consistent with previous reports. The radioligand saturation binding assay and competitive assay revealed that [123I]TZ6019 specifically bound to the human P2X7 receptor with high affinity (Ki = 6.3 ± 0.9nM). In vitro autoradiography quantification with brain slices collected from 9-month old P301S tau transgenic mice along with wild type controls, revealed higher binding of [123I]TZ6019 (35% increase) in the brain of P301S transgenic mice (n = 3, p = 0.04) compared to wild type controls. The immunofluorescence microscopy confirmed that expression of P2X7 receptor was colocalized with astrocytes in the tauopathy P301S transgenic mice. [123I]TZ6019 has specific binding for P2X7 receptor and has great potential to be a radiotracer for screening new compounds and quantifying expression of P2X7 receptor in neuroinflammation related diseases.

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 (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.

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. Acute tissue injury is attenuated in the C1KOs and this correlates with an absence of the rise in tissue reactive oxygen species (ROS) that is seen in WT mice. Infiltration of injured tissue by inflammatory cells was comparable between WT and C1KOs. Absence of CLIC1 increased PMA-induced superoxide production by isolated peritoneal neutrophils but dramatically decreased PMA-induced superoxide production by peritoneal macrophages. CLIC1 is expressed in both neutrophils and macrophages in a peripheral pattern consistent with either plasma membrane or the cortical cytoskeleton in resting cells and redistributes away from the periphery following PMA stimulation in both cell types. Absence of CLIC1 had no effect on redistribution or dephosphorylation of Ezrin/ERM cytoskeleton in macrophages. Plasma membrane chloride conductance is altered in the absence of CLIC1, but not in a way that would be expected to block superoxide production. NADPH oxidase redistributes from an intracellular compartment to the plasma membrane when WT macrophages are stimulated to produce superoxide and this redistribution fails to occur in C1KO macrophages. We conclude that the role of CLIC1 in macrophage superoxide production is to support redistribution of NADPH oxidase to the plasma membrane, and not through major effects on ERM cytoskeleton or by acting as a plasma membrane chloride channel.

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. METHODS: [C]GSK1482160 was synthesized with high specific activity and high radiochemical purity. Radioligand saturation and competition binding assays were performed for [C]GSK1482160 using HEK293-hP2X7R living cells. Micro-PET studies were carried out in nonhuman primates. In vitro autoradiography and immunohistochemistry studies were then carried out to evaluate tracer uptake and P2X7 expression in experimental autoimmune encephalomyelitis (EAE) rat lumbar spinal cord at EAE-peak and EAE-remitting stages compared with sham rats. RESULTS: [C]GSK1482160 binds to HEK293-hP2X7R living cells with high binding affinity (Kd=5.09±0.98 nmol/l, Ki=2.63±0.6 nmol/l). Micro-PET studies showed high tracer retention and a homogeneous distribution in the brain of nonhuman primates. In the EAE rat model, tracer uptake of [C]GSK1482160 in rat lumbar spinal cord was the highest at the EAE-peak stage (277.74±79.74 PSL/mm), followed by the EAE-remitting stage(149.00±54.14 PSL/mm) and sham (66.37±1.48 PSL/mm). The tracer uptake correlated strongly with P2X7-positive cell counts, activated microglia numbers, and disease severity. CONCLUSION: We conclude that [C]GSK1482160 has the potential for application in monitoring neuroinflammation.

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(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus.

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. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes.

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. We have since demonstrated that A3AR agonists have potent analgesic actions in preclinical rodent models of neuropathic pain and that A3AR analgesia is independent of adenosine A1 or A2A unwanted effects. Herein, we explored the contribution of the GABA inhibitory system to A3AR-mediated analgesia using well-characterized mouse and rat models of chronic constriction injury (CCI)-induced neuropathic pain. The deregulation of GABA signaling in pathophysiological pain states is well established: GABA signaling can be hampered by a reduction in extracellular GABA synthesis by GAD65 and enhanced extracellular GABA reuptake via the GABA transporter, GAT-1. In neuropathic pain, GABAAR-mediated signaling can be further disrupted by the loss of the KCC2 chloride anion gradient. Here, we demonstrate that A3AR agonists (IB-MECA and MRS5698) reverse neuropathic pain via a spinal mechanism of action that modulates GABA activity. Spinal administration of the GABAA antagonist, bicuculline, disrupted A3AR-mediated analgesia. Furthermore, A3AR-mediated analgesia was associated with reductions in CCI-related GAD65 and GAT-1 serine dephosphorylation as well as an enhancement of KCC2 serine phosphorylation and activity. Our results suggest that A3AR-mediated reversal of neuropathic pain increases modulation of GABA inhibitory neurotransmission both directly and indirectly through protection of KCC2 function, underscoring the unique utility of A3AR agonists in chronic pain.

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. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca(2+) flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca(2+) component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca(2+) to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca(2+) current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca(2+) through the channel.

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. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current-voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.

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. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of how P2X receptors conduct the inward flux of Na(+) and Ca(2+) in response to binding by ATP. However, this work has relied heavily on results from current recordings of P2X receptors altered by site-directed mutagenesis. In the absence of a 3-dimensional channel structure, this prior work provided only a vague and indirect appreciation of the relationship between structure, ion selectivity and flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor has thus proved a significant boon, and has provided an important opportunity to overview the amassed functional data in the context of a working 3-dimensional model of a P2X receptor. In this paper, we will attempt to reconcile the existing functional data regarding ion permeation through P2X receptors with the available crystal structure data, highlighting areas of concordance and discordance as appropriate.

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. Ca(2+) entry through P2X4 receptors is known to trigger downstream signaling pathways in microglia. Our experiments show that the lateral portals could present a novel target for drugs in the treatment of microglia-associated disease including neuropathic pain.

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. Using simultaneous recordings of membrane current and fura-2 fluorescence to measure the fractional Ca(2+) current of whole-cell currents evoked through wild-type and mutant TRPV1, we investigated a possible link between the mechanisms underlying Ca(2+) permeation and the Ca(2+)-dependent effect on conductance. Surprisingly, we found no evidence of a structural correlation, and observed that the substitution of amino acids known to regulate Ca(2+) permeability had little effect on the ability for Ca(2+) to decrease TRPV1 conductance. However, we did observe that the Ca(2+)-dependent effect on conductance was not diminished by negative hyperpolarisation for a mutant receptor with severely impaired Ca(2+) permeability, TRPV1-D646N/E648Q/E651Q. This would be consistent with the idea that Ca(2+) reduces conductance by interacting with an intra-pore binding site, and that negative hyperpolarization reduces occupancy of this site by speeding the exit of Ca(2+) into the cell. Taken together, our data show that in addition to directly and indirectly regulating channel gating, Ca(2+) also directly reduces the conductance of TRPV1. Surprisingly, the mechanism underlying this Ca(2+)-dependent effect on conductance is largely independent of mechanisms governing Ca(2+) permeability.

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. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.

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. It was demonstrated previously that inhibiting pannexin 1 with carbenoxolone inhibits hypotonically induced ATP release from human erythrocytes. Here we demonstrate that three structurally dissimilar compounds known to inhibit pannexin 1 prevent ATP release in response to lowered O(2) tension but not to iloprost-induced ATP release. These results suggest that pannexin 1 is the conduit for ATP release from erythrocytes in response to lowered O(2) tension. However, the identity of the conduit for iloprost-induced ATP release remains unknown.