Control of P2X(2) Channel Permeability by the Cytosolic Domain

The Journal of General Physiology. Aug, 2002  |  Pubmed ID: 12149275

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na(+)-selective I(1) state to the I(2) state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X(2) (rP2X(2)) channels does not occur at mouse P2X(2) (mP2X(2)) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X(2) and mP2X(2) channels have a pore diameter of approximately 11 A in the I(1) state, but the transition to the I(2) state increases the rP2X(2) diameter by at least 3 A. The I(1) to I(2) transition occurs with a rate constant of approximately 0.5 s(-1). The data focus attention on specific residues of P2X(2) channel cytoplasmic domains as determinants of permeation in a state-specific manner.

ATP Modulation of Excitatory Synapses Onto Interneurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2003  |  Pubmed ID: 12917379

Inhibitory interneurons play important roles in neuronal circuits, but the synaptic mechanisms that regulate excitatory input onto interneurons remain to be fully understood. We show that ATP-gated presynaptic P2X2 channels facilitate excitatory transmission onto stratum radiatum interneurons but not onto CA1 pyramidal neurons. ATP released endogenously during carbachol-induced oscillations facilitates excitatory synapses onto interneurons. Overall, these data provide evidence for the molecular identity, synaptic function, and interneuron synapse specificity of a presynaptic neurotransmitter-gated cation channel. The findings highlight a novel form of presynaptic facilitation for hippocampal interneurons and suggest a role for extracellular ATP in neuronal networks.

ATP-gated P2X Channels

Current Biology : CB. Jan, 2004  |  Pubmed ID: 14711422

Contribution of Calcium Ions to P2X Channel Responses

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2004  |  Pubmed ID: 15056721

Ca2+ entry through transmitter-gated cation channels, including ATP-gated P2X channels, contributes to an array of physiological processes in excitable and non-excitable cells, but the absolute amount of Ca2+ flowing through P2X channels is unknown. Here we address the issue of precisely how much Ca2+ flows through P2X channels and report the finding that the ATP-gated P2X channel family has remarkably high Ca2+ flux compared with other channels gated by the transmitters ACh, serotonin, protons, and glutamate. Several homomeric and heteromeric P2X channels display fractional Ca2+ currents equivalent to NMDA channels, which hitherto have been thought of as the largest source of transmitter-activated Ca2+ flux. We further suggest that NMDA and P2X channels may use different mechanisms to promote Ca2+ flux across membranes. We find that mutating three critical polar amino acids decreases the Ca2+ flux of P2X2 receptors, suggesting that these residues cluster to form a novel type of Ca2+ selectivity region within the pore. Overall, our data identify P2X channels as a large source of transmitter-activated Ca2+ influx at resting membrane potentials and support the hypothesis that polar amino acids contribute to Ca2+ selection in an ATP-gated ion channel.

ATP Excites Interneurons and Astrocytes to Increase Synaptic Inhibition in Neuronal Networks

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2004  |  Pubmed ID: 15456834

We investigated the role of extracellular ATP at astrocytes and inhibitory GABAergic interneurons in the stratum radiatum area of the mouse hippocampus. We show that exogenously applied ATP increased astrocyte intracellular Ca2+ levels and depolarized all calbindinand calretinin-positive interneurons in the stratum radiatum region of mouse hippocampus, leading to action potential firing and enhanced synaptic inhibition onto the postsynaptic targets of interneurons. Electrophysiological, pharmacological, and immunostaining studies suggested that the effect of ATP on interneurons was mediated by P2Y1 receptors, and that the depolarization of interneurons was caused by the concomitant reduction and activation of potassium and nonselective cationic conductances, respectively. Electrical stimulation of the Schaffer collaterals and perforant path, as well as local stimulation within the stratum radiatum, evoked increases in intracellular Ca2+ in astrocytes. Facilitation of GABAergic IPSCs onto interneurons also occurred during electrical stimulation. Both the stimulation-evoked increases in astrocyte Ca2+ levels and facilitation of GABAergic IPSCs were sensitive to antagonists of P2Y1 receptors and mimicked by exogenous P2Y1 receptor agonists, suggesting that endogenously released ATP can activate P2Y receptors on both astrocytes and interneurons. Overall, our data are consistent with the hypothesis that ATP released from neurons and astrocytes acts on P2Y1 receptors to excite interneurons, resulting in increased synaptic inhibition within intact hippocampal circuits.

Time-resolved Measurement of State-specific P2X2 Ion Channel Cytosolic Gating Motions

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2004  |  Pubmed ID: 15548662

ATP-gated P2X2 channels undergo permeability changes through a process that is incompletely understood. In the present study, we used fluorescence resonance energy transfer (FRET) and electrophysiology to measure cytosolic gating motions in P2X2 channels as they enter a state with increased permeability. P2X2 channels underwent permeability changes with a time course that was similar to decreases in FRET between cyan fluorescent protein and yellow fluorescent protein attached to the cytosolic domain of P2X2 channels. Wild-type and mutant channels that did not undergo permeability changes also did not show evidence of cytosolic gating motions. Moreover, immobilizing the cytosolic domain by tethering it to the plasma membrane prevented the switch in permeability and impaired the cytosolic gating motions. Both of these phenotypes were restored when the immobilizing tether was cleaved. The data provide a time-resolved measure of state-specific gating motions and suggest how a cytosolic domain may control ion channel permeability.

Contribution of Transmembrane Regions to ATP-gated P2X2 Channel Permeability Dynamics

The Journal of Biological Chemistry. Feb, 2005  |  Pubmed ID: 15556949

ATP-gated P2X(2) channels undergo activation-dependent permeability increases as they proceed from the selective I(1) state to the I(2) state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X(2) channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X(2) channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I(1) state to the dilated I(2) state. Initial channel opening to the I(1) state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I(2) state. Ten residues appeared to perturb only the I(1)-I(2) transition (Phe(31), Arg(34), Gln(37), Lys(53), Ile(328), Ile(332), Ser(340), Gly(342), Trp(350), Leu(352)). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X(2) channels.

An Angstrom Scale Interaction Between Plasma Membrane ATP-gated P2X2 and Alpha4beta2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2005  |  Pubmed ID: 16033901

Structurally distinct nicotinic and P2X channels interact functionally, such that coactivation results in cross-inhibition of one or both channel types. It is hypothesized, but not yet proven, that nicotinic and P2X channels interact at the plasma membrane. Here, we show that plasma membrane alpha4beta2 nicotinic and P2X2 channels form a molecular scale partnership and also influence each other when coactivated, resulting in nonadditive cross-inhibitory responses. Total internal reflection fluorescence and fluorescence resonance energy transfer microscopy between fluorescently labeled P2X2 and alpha4beta2 nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X2 and alpha4beta2 channels may form a dimer, with the channels approximately 80 A apart. The measurements also show that P2X2 subunits interact specifically and robustly with the beta2 subunits in alpha4beta2 channels. The data provide direct evidence for the close spatial apposition of full-length P2X2 and alpha4beta2 channels within 100 nm of the plasma membrane of living cells.

P2X Receptors As Cell-surface ATP Sensors in Health and Disease

Nature. Aug, 2006  |  Pubmed ID: 16885977

P2X receptors are membrane ion channels activated by the binding of extracellular adenosine triphosphate (ATP). For years their functional significance was consigned to distant regions of the autonomic nervous system, but recent work indicates several further key roles, such as afferent signalling, chronic pain, and in autocrine loops of endothelial and epithelial cells. P2X receptors have a molecular architecture distinct from other ion channel protein families, and have several unique functional properties.

Two Forms of Single-vesicle Astrocyte Exocytosis Imaged with Total Internal Reflection Fluorescence Microscopy

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2007  |  Pubmed ID: 17360502

Transmitters such as glutamate and ATP are released from brain astrocytes. Several pathways for their release have been proposed, including exocytosis. In the present study we sought to measure exocytosis from astrocytes with single vesicle imaging methods using synaptopHlourin (SpH) as an optical reporter. We imaged single SpH-laden vesicles with total internal reflection fluorescence (TIRF) microscopy. We observed spontaneous, as well as evoked, single-vesicle exocytosis events. Analysis of the kinetics and spatial spread associated with these events indicated two discernible forms of single vesicle exocytosis. One form, constituting approximately 40% of the spontaneous events, was akin to kiss-and-run vesicle fusion and captured a mobile proton buffer from the extracellular medium. The other form seems to represent full vesicle fusion, constitutes approximately 60% of the spontaneous events, and is associated with complete mixing of the vesicle and plasma membranes. Activation of calcium-mobilizing receptors on the astrocyte surface selected between the different forms of exocytosis. These data provide evidence for two forms of simultaneously occurring single-vesicle exocytosis events in astrocytes, and also suggest that SpH imaging and TIRF microscopy is useful to study the mechanisms of astrocyte transmitter release.

Vesicular ATP is the Predominant Cause of Intercellular Calcium Waves in Astrocytes

The Journal of General Physiology. Jun, 2007  |  Pubmed ID: 17504911

Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between approximately 100 and 250 microm, and approximately 15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.

Tracking Transmitter-gated P2X Cation Channel Activation in Vitro and in Vivo

Nature Methods. Jan, 2008  |  Pubmed ID: 18084300

We present a noninvasive approach to track activation of ATP-gated P2X receptors and potentially other transmitter-gated cation channels that show calcium fluxes. We genetically engineered rat P2X receptors to carry calcium sensors near the channel pore and tested this as a reporter for P2X(2) receptor opening. The method has several advantages over previous attempts to image P2X channel activation by fluorescence resonance energy transfer (FRET): notably, it reports channel opening rather than a conformation change in the receptor protein. Our FRET-based imaging approach can be used as a general method to track, in real time, the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in living neurons in vitro and in vivo. This approach should help to determine when, where and how different receptors are activated during physiological processes.

Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-mediated Slow Inward Currents in Pyramidal Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2008  |  Pubmed ID: 18579739

Astrocytes display excitability in the form of intracellular calcium concentration ([Ca(2+)](i)) increases, but the signaling impact of these for neurons remains debated and controversial. A key unresolved issue is whether astrocyte [Ca(2+)](i) elevations impact neurons or not. Here we report that in the CA1 region of the hippocampus, agonists of native P2Y(1) and PAR-1 receptors, which are preferentially expressed in astrocytes, equally elevated [Ca(2+)](i) levels without affecting the passive membrane properties of pyramidal neurons. However, under conditions chosen to isolate NMDA receptor responses, we found that activation of PAR-1 receptors led to the appearance of NMDA receptor-mediated slow inward currents (SICs) in pyramidal neurons. In stark contrast, activation of P2Y(1) receptors was ineffective in this regard. The PAR-1 receptor-mediated increased SICs were abolished by several strategies that selectively impaired astrocyte [Ca(2+)](i) excitability and function. Our studies therefore indicate that evoked astrocyte [Ca(2+)](i) transients are not a binary signal for interactions with neurons, and that astrocytes result in neuronal NMDA receptor-mediated SICs only when appropriately excited. The data thus provide a basis to rationalize recent contradictory data on astrocyte-neuron interactions.

Patch-clamp Coordinated Spectroscopy Shows P2X2 Receptor Permeability Dynamics Require Cytosolic Domain Rearrangements but Not Panx-1 Channels

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2008  |  Pubmed ID: 18689682

ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I(2) state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I(2) state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X(2) receptor I(2) state. However, P2X(2) receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins.

Tunable Calcium Current Through TRPV1 Receptor Channels

The Journal of Biological Chemistry. Nov, 2008  |  Pubmed ID: 18775990

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. Using site-directed mutagenesis, we discovered that the smaller Pf% was due to protonation of three acidic amino acids (Asp646, Glu648, and Glu651) that are located in the mouth of the pore. Thus, in keeping with recent reports of time-dependent changes in the ionic permeability of some ligand-gated ion channels, we now show for the first time that the physiologically important Ca2+ current of the TRPV1 receptor is also dynamic and depends on the mode of activation. This current is significantly smaller when the receptor is activated by a change in pH, owing to atomic scale interactions of H+ and Ca2+ with the fixed negative charge of side chains in the pore.

Regulation of P2X2 Receptors by the Neuronal Calcium Sensor VILIP1

Science Signaling. 2008  |  Pubmed ID: 18922787

Extracellular adenosine triphosphate (ATP) activates P2X receptors, which are involved in diverse physiological functions. Using a proteomic approach, we identified the neuronal calcium sensor VILIP1 as interacting with P2X2 receptors. We found that VILIP1 forms a signaling complex in vitro and in vivo with P2X2 receptors and regulates P2X2 receptor sensitivity to ATP, peak response, surface expression, and diffusion. VILIP1 constitutively binds to P2X2 receptors and displays enhanced interactions in an activation- and calcium-dependent manner owing to exposure of its binding segment in P2X2 receptors. VILIP1-P2X2 interactions are also enhanced in hippocampal neurons during conditions of action potential firing known to trigger P2X2 receptor activation. Our data thus reveal a previously unrecognized function for the neuronal calcium sensor protein VILIP1 and a mechanism for regulation of ATP-dependent P2X receptor signaling by neuronal calcium sensors.

Molecular Shape, Architecture, and Size of P2X4 Receptors Determined Using Fluorescence Resonance Energy Transfer and Electron Microscopy

The Journal of Biological Chemistry. Sep, 2008  |  Pubmed ID: 18635539

P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X(4) receptors was 5.6 nm. Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin. We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain. In this structure, the distance between the centers of the gold particles was 6.1 nm, which closely matches FRET data. Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.

ATP-gated P2X Cation-channels

Neuropharmacology. Jan, 2009  |  Pubmed ID: 18657557

P2X receptors are ATP-gated cation channels with important roles in diverse pathophysiological processes. Substantial progress has been made in the last few years with the discovery of both subunit selective antagonists and modulators. The purpose of this brief review is to summarize the advances in the pharmacology of P2X receptors, with key properties presented in an easy to access format. Ligand-gated ion channels consist of three families in mammals; the ionotropic glutamate receptors, the Cys-loop receptors (for GABA, ACh, glycine and serotonin) and the P2X receptors for ATP. The first two of these are considered in articles accompanying this Special Issue. Here we consider the pharmacological properties of P2X receptors. We do not present a detailed discussion of P2X receptor physiological roles or structure-function studies. Moreover, the pharmacological basis for discriminating between the main subtypes of P2X receptor and their nomenclature has been published by the Nomenclature Committee of the International Union of Pharmacology (NC-IUPHAR) P2X Receptor Subcommittee, and so these aspects are not revisited here. Instead in this brief article we seek to present a summary of the pharmacology of recombinant homomeric and heteromeric P2X receptors, with particular emphasis on new antagonists. In this article we have tried to present as much information as possible in two tables in the hope this will be useful as a day-to-day resource, and also because an excellent and detailed review has recently been published.

The Double Life of ATP

Scientific American. Dec, 2009  |  Pubmed ID: 20058644

ATP-gated P2X Receptors on Excitatory Nerve Terminals Onto Interneurons Initiate a Form of Asynchronous Glutamate Release

Neuropharmacology. Jan, 2009  |  Pubmed ID: 18601937

Previous work has shown that ATP-gated P2X2 receptors are expressed in excitatory nerve terminals onto stratum radiatum interneurons in the mouse hippocampal CA1 region. At these synapses receptor activation results in calcium-dependent facilitation of miniature and spontaneous EPSC frequency. In this study I determined if activation of presynaptic P2X receptors produces these effects by utilizing the vesicles underlying action potential dependent release. Brief trains of electrical stimuli caused short-term synaptic depression of excitatory synapses onto interneurons, in a manner consistent with depletion of the readily releasable pool of vesicles. P2X receptor activation increased the frequency of spontaneous EPSCs, but unexpectedly evoked little effect on synaptic depression. This suggests that P2X receptor activation does not markedly draw on the vesicles underlying action potential dependent glutamate release. However asynchronous EPSCs were increased following synaptic depression and a component of these appeared to be initiated by endogenously released ATP acting on presynaptic P2X receptors. Unexpectedly, the data suggest P2X receptor activation initiates a form of asynchronous glutamate release, rather than detectably affecting the vesicles underlying action potential evoked release.

Gated Access to the Pore of a P2X Receptor: Structural Implications for Closed-open Transitions

The Journal of Biological Chemistry. Mar, 2010  |  Pubmed ID: 20093367

P2X receptors are ligand-gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor directly reveals that the ion-conducting pathway is formed by three transmembrane domain 2 (TM2) alpha-helices, each being provided by the three subunits of the trimer. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In this study, we quantified gated access to Cd(2+) at substituted cysteines in TM2 of P2X2 receptors in the open and closed states. Our data for the closed state are consistent with the zebrafish P2X4.1 structure, with isoleucines and threonines (Ile-332 and Thr-336) positioned one helical turn apart lining the channel wall on approach to the gate. Our data for the open state reveal gated access to deeper parts of the pore (Thr-339, Val-343, Asp-349, and Leu-353), suggesting the closed channel gate is between Thr-336 and Thr-339. We also found unexpected interactions between native Cys-348 and D349C that result in tight Cd(2+) binding deep within the intracellular vestibule in the open state. Interpreted with a P2X2 receptor structural model of the closed state, our data suggest that the channel gate opens near Thr-336/Thr-339 and is accompanied by movement of the pore-lining regions, which narrow toward the cytosolic end of TM2 in the open state. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.

P2X4 Receptors in Activated C8-B4 Cells of Cerebellar Microglial Origin

The Journal of General Physiology. Apr, 2010  |  Pubmed ID: 20231374

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 approximately 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.

A Genetically Targeted Optical Sensor to Monitor Calcium Signals in Astrocyte Processes

Nature Neuroscience. Jun, 2010  |  Pubmed ID: 20495558

Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential-mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes.

Monitoring Astrocyte Calcium Microdomains with Improved Membrane Targeted GCaMP Reporters

Neuron Glia Biology. Dec, 2010  |  Pubmed ID: 21205365

Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.

Bulk Loading of Calcium Indicator Dyes to Study Astrocyte Physiology: Key Limitations and Improvements Using Morphological Maps

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2011  |  Pubmed ID: 21697385

Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ∼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as "maps" after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.

Preferential Use of Unobstructed Lateral Portals As the Access Route to the Pore of Human ATP-gated Ion Channels (P2X Receptors)

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2011  |  Pubmed ID: 21808018

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.

Neuronal P2X2 Receptors Are Mobile ATP Sensors That Explore the Plasma Membrane when Activated

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2011  |  Pubmed ID: 22090499

ATP-gated ionotropic P2X2 receptors are widely expressed in neurons. Although the electrophysiological properties of P2X2 receptors have been extensively studied, little is known about the plasma membrane lateral mobility of P2X2 receptors or whether receptor mobility is regulated by ATP. Here we used single-molecule imaging with simultaneous whole-cell voltage-clamp recordings to track quantum dot-labeled P2X2 receptors in the dendrites of rat hippocampal neurons to explore P2X2 receptor mobility and its regulation. We find that plasma membrane P2X2 receptor lateral mobility in dendrites is heterogeneous but mostly Brownian in nature, consisting of mobile and slowly mobile receptor pools. Moreover, lateral mobility is P2X2 subunit and cell specific, is increased in an activation-dependent manner, and is regulated by cytosolic VILIP1, a calcium binding protein. Our data provide the first direct measures of P2X receptor mobility and show that P2X2 receptors are mobile ATP sensors, sampling more of the dendritic plasma membrane in response to ATP.

TRPA1 Channels Regulate Astrocyte Resting Calcium and Inhibitory Synapse Efficacy Through GAT-3

Nature Neuroscience. Jan, 2012  |  Pubmed ID: 22158513

Astrocytes contribute to the formation and function of synapses and are found throughout the brain, where they show intracellular store-mediated Ca(2+) signals. Here, using a membrane-tethered, genetically encoded calcium indicator (Lck-GCaMP3), we report the serendipitous discovery of a new type of Ca(2+) signal in rat hippocampal astrocyte-neuron cocultures. We found that Ca(2+) fluxes mediated by transient receptor potential A1 (TRPA1) channels gave rise to frequent and highly localized 'spotty' Ca(2+) microdomains near the membrane that contributed appreciably to resting Ca(2+) in astrocytes. Mechanistic evaluations in brain slices showed that decreases in astrocyte resting Ca(2+) concentrations mediated by TRPA1 channels decreased interneuron inhibitory synapse efficacy by reducing GABA transport by GAT-3, thus elevating extracellular GABA. Our data show how a transmembrane Ca(2+) source (TRPA1) targets a transporter (GAT-3) in astrocytes to regulate inhibitory synapses.

Allosteric Modulation of Ca2+ Flux in a Ligand-gated Cation Channel (P2X4) by Actions on Lateral Portals

The Journal of Biological Chemistry. Jan, 2012  |  Pubmed ID: 22219189

Human P2X receptors are a family of seven ATP-gated ion channels that transport Na+, K+, and Ca2+ across cell surface membranes. The P2X4 receptor is unique amongst 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 (Glu51) in the lateral entrance to the transmembrane pore markedly attenuates the effect of ivermectin on Ca2+ current and channel gating. Ca2+ entry through P2X4 receptors is known to trigger downstream signalling 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.