Copper ions, prion protein and Aß modulate Ca levels in central nervous system myelin in an NMDA receptor-dependent manner.

As in neurons, CNS myelin expresses N-Methyl-D-Aspartate Receptors (NMDARs) that subserve physiological roles, but have the potential to induce injury to this vital element. Using 2-photon imaging of myelinic Ca in live ex vivo mouse optic nerves, we show that Cu ions potently modulate Ca levels in an NMDAR-dependent manner. Chelating Cu in the perfusate induced a substantial increase in Ca levels, and also caused significant axo-myelinic injury. Myelinic NMDARs are shown to be regulated by cellular prion protein; only in prion protein KO optic nerves does application of NMDA + D-serine induce a large Ca increase, consistent with strong desensitization of these receptors in the presence of prion protein limiting Ca overload. Aß1-42 peptide induced a large Ca increase that was also Cu-dependent, and was blocked by NMDAR antagonism. Our results indicate that like in neurons, myelinic NMDARs permeate potentially injurious amounts of Ca, and are also potently regulated by micromolar Cu and activated by Aß1-42 peptides. These findings shed mechanistic light on the important primary white matter injury frequently observed in Alzheimer's brain.

Development and validation of a UPLC-MS/MS method for quantification of C-005, a novel third-generation EGFR TKI, and its major metabolite in plasma: Application to its first-in-patient study.

C-005 is a novel third-generation EGFR tyrosine kinase inhibitor for the treatment of non-small cell lung cancer (NSCLC). To support its clinical trial, we developed a rapid and sensitive bioanalytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique for the quantification of C-005 and its major metabolite in NSCLC patients following international bioanalytical guidelines. After a simple and quick protein precipitation step, the supernatant was injected to a Waters Acquity BEH C18 column (2.1 × 50 mm i.d., 1.7 mm), and the column was eluted with a gradient of buffer A (5 mM ammonium acetate and 0.1% formic acid in water) and buffer B (formic acid-acetonitrile (1:1000, v/v)). The eluates were subsequently detected by an AB QTRAP 5500 mass spectrometer with electrospray ionization using multiple-reaction monitoring mode. The method showed good linearity from 2.00 to 1000 ng/mL for C-005 and 1.00 to 500 ng/mL for M1. In conclusion, the validation results demonstrated the robustness of the method and its well-poised to support the first-in-patient study of C-005 in NSCLC patients.

Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist.

BACKGROUND: Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181. RESULTS: NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist). CONCLUSIONS: Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.

Disruption of NMDAR-CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model.

Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca ( 2+) channels. Building on our discovery of the interaction and regulation of Ca ( 2+) channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca ( 2+) channel. Remarkably, we also found that this region attenuated Ca ( 2+) influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2's role in excitotoxicity and neuroprotection.

Aß neurotoxicity depends on interactions between copper ions, prion protein, and N-methyl-D-aspartate receptors.

N-methyl-d-aspartate receptors (NMDARs) mediate critical CNS functions, whereas excessive activity contributes to neuronal damage. At physiological glycine concentrations, NMDAR currents recorded from cultured rodent hippocampal neurons exhibited strong desensitization in the continued presence of NMDA, thus protecting neurons from calcium overload. Reducing copper availability by specific chelators (bathocuproine disulfonate, cuprizone) induced nondesensitizing NMDAR currents even at physiologically low glycine concentrations. This effect was mimicked by, and was not additive with, genetic ablation of cellular prion protein (PrP(C)), a key copper-binding protein in the CNS. Acute ablation of PrP(C) by enzymatically cleaving its cell-surface GPI anchor yielded similar effects. Biochemical studies and electrophysiological measurements revealed that PrP(C) interacts with the NMDAR complex in a copper-dependent manner to allosterically reduce glycine affinity for the receptor. Synthetic human Aß(1-42) (10 nM-5 µM) produced an identical effect that could be mitigated by addition of excess copper ions or NMDAR blockers. Taken together, Aß(1-42), copper chelators, or PrP(C) inactivation all enhance the activity of glycine at the NMDAR, giving rise to pathologically large nondesensitizing steady-state NMDAR currents and neurotoxicity. We propose a physiological role for PrP(C), one that limits excessive NMDAR activity that might otherwise promote neuronal damage. In addition, we provide a unifying molecular mechanism whereby toxic species of Aß(1-42) might mediate neuronal and synaptic injury, at least in part, by disrupting the normal copper-mediated, PrP(C)-dependent inhibition of excessive activity of this highly calcium-permeable glutamate receptor.

Copper-dependent regulation of NMDA receptors by cellular prion protein: implications for neurodegenerative disorders.

N-Methyl-D-aspartate (NMDA) receptors mediate a wide range of important nervous system functions. Conversely, excessive NMDA receptor activity leads to cytotoxic calcium overload and neuronal damage in a wide variety of CNS disorders. It is well established that NMDA receptors are tightly regulated by a number of cell signalling pathways. Recently, it has been shown that NMDA receptor activity is modulated by cellular prion protein (PrP(C)) in a copper-dependent manner. Here we give an overview of the current state of knowledge concerning the novel concept of potent modulation of this receptor's kinetics by copper ions, and the interplay between NMDA receptors and PrP(C) in the context of neurological diseases such as Alzheimer's disease, epilepsy, pain and depression.

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands.

BACKGROUND: Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity. RESULTS: Novel compounds were characterized in radioligand binding assays and in vitro functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compounds on transient expressed human T-type calcium channels were examined in tsA-201 cells using standard whole-cell voltage clamp techniques, and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, intraperitoneally) assessed in the formalin model. A series of compounds were synthesized and evaluated for channel and receptor activity. Compound NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compounds completely inhibited peak Cav3.2 currents with IC50 values in the low micromolar range. All compounds mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response. CONCLUSIONS: Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects in vivo. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.

Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2).

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.

The Cavß subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels.

It is well established that the auxiliary Cavß subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavß subunit increased membrane expression of Cav1.2 channels by preventing the entry of the channels into the endoplasmic reticulum-associated protein degradation (ERAD) complex. Without Cavß, Cav1.2 channels underwent robust ubiquitination by the RFP2 ubiquitin ligase and interacted with the ERAD complex proteins derlin-1 and p97, culminating in targeting of the channels to the proteasome for degradation. On treatment with the proteasomal inhibitor MG132, Cavß-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavß interfered with ubiquitination and targeting of the channel to the ERAD complex, thereby facilitating export from the endoplasmic reticulum and promoting expression on the cell surface. Thus, Cavßß regulates the ubiquitination and stability of the calcium channel complex.

Benzodiazepine modulation of the rat GABAA receptor α4ß3γ2L subtype expressed in Xenopus oocytes.

The effects of benzodiazepines on GABA(A) receptors are dependent largely on the particular α subunit isoform that is present in the receptor pentamer. The inclusion of either the α4 or α6 subunit is generally thought to render the receptor insensitive to classical benzodiazepines. We expressed the rat α4ß3γ2L subtype in Xenopus oocytes and observed that both diazepam and flunitrazepam significantly potentiated GABA-gated currents. This potentiation occurred at nanomolar concentrations similar to those seen at the most abundant "diazepam-sensitive" receptor i.e., the α1ß2γ2 subtype. In the α4ß3γ2L receptor, the effects of diazepam and flunitrazepam were inhibited by nanomolar concentrations of the benzodiazepine site antagonists, Ro15-1788 and ZK93426. The presence of the ß3 subunit appears to be important for this modulation since diazepam did not affect GABA responses mediated by recombinant α4ß1γ2L or α4ß2γ2L receptors. Interestingly, when the α4ß3γ2L receptor was expressed in HEK293 cells, diazepam and flunitrazepam displaced the relatively non-selective benzodiazepine site ligand, [(3)H]Ro15-4513, only at high concentrations (>10 µM) demonstrating a lack of high affinity binding for these classical benzodiazepines. Functional studies of the cell-expressed receptors using whole cell recording techniques showed that neither diazepam nor flunitrazepam potentiated GABA-evoked currents although currents were enhanced by nanomolar concentrations of Ro15-4513. These results suggest that the observed benzodiazepine modulation of the α4ß3γ2L subtype depends on the expression system used and may be specific for expression in Xenopus oocytes.

Heterodimerization of ORL1 and opioid receptors and its consequences for N-type calcium channel regulation.

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, mu-, delta-, and kappa-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The mu-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of mu-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.

CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels.

Monocyte chemoattractant protein-1 (MCP-1) is a cytokine known to be involved in the recruitment of monocytes to sites of injury. MCP-1 activates the chemokine (C-C motif) receptor 2 (CCR2), a seven-transmembrane helix G protein-coupled receptor that has been implicated in inflammatory pain responses. Here we show that MCP-1 mediates activation of the CCR2 receptor and inhibits coexpressed N-type calcium channels in tsA-201 cells via a voltage-dependent pathway. Moreover, MCP-1 inhibits Ca(v)3.2 calcium channels, but not other members of the Cav3 calcium channel family, with nanomolar affinity. Unlike in N-type channels, this modulation does not require CCR2 receptor activation and seems to involve a direct action of the ligand on the channel. Whole-cell T-type calcium currents in acutely dissociated dorsal root ganglia neurons are effectively inhibited by MCP-1, consistent with the notion that these cells express Ca(v)3.2. The effects of MCP-1 were eliminated by heat denaturation. Furthermore, they were sensitive to the application of the divalent metal ion chelator diethylenetriaminepentaacetic acid, suggesting the possibility that metal ions may act as a cofactor. Finally, small organic CCR2 receptor antagonists inhibit Ca(v)3.2 and other members of the T-type channel family with micromolar affinity. Our findings provide novel avenues for the design of small organic inhibitors of T-type calcium channels for the treatment of pain and other T-type channel linked disorders.

Bioactive contaminants leach from disposable laboratory plasticware.

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

Atomic force microscopy reveals the stoichiometry and subunit arrangement of the alpha4beta3delta GABA(A) receptor.

The GABA(A) receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains alpha(1)-, beta(2)-, and gamma(2)-subunits in the arrangement alphabetaalphagammabeta, counter-clockwise around the pore. GABA(A) receptors containing delta-instead of gamma-subunits, although a minor component of the total receptor population, have interesting properties, such as an extrasynaptic location, high sensitivity to GABA, and potential association with conditions such as epilepsy. They are therefore attractive targets for drug development. Here we addressed the subunit arrangement within the alpha(4)beta(3)delta form of the receptor. Different epitope tags were engineered onto the three subunits, and complexes between receptors and anti-epitope antibodies were imaged by atomic force microscopy. Determination of the numbers of receptors doubly decorated by each of the three antibodies revealed a subunit stoichiometry of 2alpha:2beta:1delta. The distributions of angles between pairs of antibodies against the alpha- and beta-subunits both had peaks at around 144 degrees , indicating that these pairs of subunits were nonadjacent. Decoration of the receptor with ligands that bind to the extracellular domain (i.e., the lectin concanavalin A and an antibody that recognizes the beta-subunit N-terminal sequence) showed that the receptor preferentially binds to the mica extracellular face down. Given this orientation, the geometry of complexes of receptors with both an antibody against the delta-subunit and Fab fragments against the alpha-subunits indicates a predominant subunit arrangement of alphabetaalphadeltabeta, counter-clockwise around the pore when viewed from the extracellular space.

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity.

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.