Browsing by Author "You, Haitao"
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Item Open Access Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist(BioMed Central Ltd., 2013-07-01) You, Haitao; Gadotti, Vinícius Maria; Petrov, Ravil R.; Berger, N. Daniel; Diaz, Philippe J.; Zamponi, Gerald W.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.Item Open Access Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist(BioMed Central, 2013-07-01) Gadotti, Vinicius M; You, Haitao; Petrov, Ravil R; Berger, N Daniel; Diaz, Philippe; Zamponi, Gerald WItem Open Access The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels(Nature, 2011-02) Altier, Christophe; You, Haitao; Chen, Lina; Walcher, Jan; Hermosilla, Tamara; García-Caballero, Agustín; Simms, Brent A.; Tedford, H. William; Zamponi, Gerald W.It is well established that the auxiliary Cavβ subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavβ 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 Cavβ, 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, Cavβ-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavβ 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, Cavββ regulates the ubiquitination and stability of the calcium channel complex.Item Open Access CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels(The American Society for Pharmacology and Experimental Therapeutics, 2010-02-01) You, Haitao; Altier, Christophe; Zamponi, Gerald W.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.Item Open Access Copper ions, prion protein and Aβ modulate Ca levels in central nervous system myelin in an NMDA receptor-dependent manner(2022-07-26) Tsutsui, Shigeki; Morgan, Megan; Tedford, Hugo; You, Haitao; Zamponi, Gerald W.; Stys, Peter K.Abstract 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. Aβ1-42 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 Aβ1-42 peptides. These findings shed mechanistic light on the important primary white matter injury frequently observed in Alzheimer's brain.Item Open Access Copper-dependent regulation of NMDA receptors by cellular prion protein: implications for neurodegenerative disorders(The Physiological Society, 2012-02-06) Stys, Peter K.; You, Haitao; Zamponi, Gerald W.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.Item Open Access Disruption of NMDAR-CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model(Landes Bioscience, 2012-01-01) Pan, Rui; You, Haitao; Brustovetsky, Tatiana; Khanna, Rajesh; Brittain, Joel M.; Brustovetsky, Nickolay N.; Zamponi, Gerald W.; Lee, WeihuaCollapsin 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.Item Open Access Heterodimerization of ORL1 and opioid receptors and its consequences for N-type calcium channel regulation(The American Society for Biochemistry and Molecular Biology, Inc., 2010-01-08) You, Haitao; Hameed, Shahid; Altier, Christophe; Mezghrani, Alexandre; Bourinet, Emmanuel; Evans, Rhian M.; Zamponi, Gerald W.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.Item Open Access Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2)(The American Society for Biochemistry and Molecular Biology, Inc., 2011-08-09) Brittain, Joel M.; Chen, Liang; Wilson, Sarah M.; Brustovetsky, Tatiana; Gao, Xiang; Ashpole, Nicole M.; Molosh, Andrei I.; You, Haitao; Hudmon, Andy; Shekhar, Anantha S.; White, Fletcher A.; Zamponi, Gerald W.; Brustovetsky, Nickolay N.; Chen, Jinhui; Khanna, RajeshNeurological 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.