Browsing by Author "Chen, Lina"

Now showing 1 - 13 of 13
  • Thumbnail Image
    ItemOpen Access
    Cav3.2 calcium channel interactions with the epithelial sodium channel ENaC
    (2019-02-08) Garcia-Caballero, Agustin; Gandini, Maria A; Huang, Shuo; Chen, Lina; Souza, Ivana A; Dang, Yan L; Stutts, M. J; Zamponi, Gerald W
    Abstract This study describes the functional interaction between Cav3.2 calcium channels and the Epithelial Sodium Channel (ENaC). β-ENaC subunits showed overlapping expression with endogenous Cav3.2 calcium channels in the thalamus and hypothalamus as detected by immunostaining. Moreover, β- and γ-ENaC subunits could be co-immunoprecipitated with Cav3.2 calcium channels from brain lysates, dorsal horn and lumbar dorsal root ganglia. Mutation of a cluster of lysines present in the intracellular N-terminus region of β-ENaC (K4R/ K5R/ K9R/ K16R/ K23R) reduced interactions with Cav3.2 calcium channels. Αβγ-ENaC channels enhanced Cav3.2 calcium channel trafficking to the plasma membrane in tsA-201 cells. This effect was reciprocal such that Cav3.2 channel expression also enhanced β-ENaC trafficking to the cell surface. T-type current density was increased when fully assembled αβγ-ENaC channels were transiently expressed in CAD cells, a neuronal derived cell line. Altogether, these findings reveal ENaC as an interactor and potential regulator of Cav3.2 calcium channels expressed in neuronal tissues.
  • Thumbnail Image
    ItemOpen 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.
  • Thumbnail Image
    ItemOpen Access
    A cell-permeant peptide corresponding to the cUBP domain of USP5 reverses inflammatory and neuropathic pain
    (Sage Publishing, 2016-01-01) García-Caballero, Agustín; Gadotti, Vinícius Maria; Chen, Lina; Zamponi, Gerald W.
    Cav3.2 T-type calcium currents in primary afferents are enhanced in various painful pathological conditions, whereas inhibiting Cav3.2 activity or expression offers a strategy for combating the development of pain hypersensitivity. We have shown that Cav3.2 channel surface density is strongly regulated by the ubiquitination machinery and we identified the deubiquitinase USP5 as a Cav3.2 channel interacting protein and regulator of its cell surface expression. We also reported that USP5 is upregulated in chronic pain conditions. Conversely, preventing its binding to the channel in vivo mediates analgesia in inflammatory and neuropathic pain models.
  • Thumbnail Image
    ItemOpen Access
    D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry
    (Elsevier, 2008-05-22) Kisilevsky, Alexandra E.; Mulligan, Sean J.; Altier, Christophe; Iftinca, Mircea C.; Varela, Diego L.; Tai, Chao; Chen, Lina; Hameed, Shahid; Hamid, Jawed; MacVicar, Brian Archibald; Zamponi, Gerald W.
    Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.
  • Thumbnail Image
    ItemOpen Access
    Differential modulation of NMDA and AMPA receptors by cellular prion protein and copper ions
    (2018-10-25) Huang, Sun; Chen, Lina; Bladen, Chris; Stys, Peter K; Zamponi, Gerald W
    Abstract N-Methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are two major types of ionotropic glutamate receptors involved in synaptic transmission. However, excessive activity of these receptors can be cytotoxic and thus their function must be precisely controlled. We have previously reported that NMDA receptor activity is dysregulated following genetic knockout of cellular prion protein (PrPC), and that PrPC regulation of NMDA receptors is copper-dependent. Here, we employed electrophysiological methods to study NMDAR and AMPAR currents of cultured hippocampal neurons from PrPC overexpresser mice. We show that NMDA receptor current amplitude and kinetics are differentially modulated by overexpression of human or mouse PrPC. By contrast, AMPA receptor activity was unaffected. Nonetheless, AMPA receptor activity was modulated by copper ions in a manner similar to what we previously reported for NMDA receptors. Taken together, our findings reveal that AMPA and NMDA receptors are differentially regulated by PrPC, but share common modulation by copper ions.
  • Thumbnail Image
    ItemOpen Access
    Inhibitory insula-ACC projections modulate affective but not sensory aspects of neuropathic pain
    (2023-08-21) Alonso-Matielo, Heloísa; Zhang, Zizhen; Gambeta, Eder; Huang, Junting; Chen, Lina; de Melo, Gabriel O.; Dale, Camila S.; Zamponi, Gerald W.
    Abstract The insula and anterior cingulate cortex (ACC) are brain regions that undergo structural and functional reorganization in neuropathic pain states. Here, we aimed to study inhibitory parvalbumin positive (PV+) posterior insula (pIC) to posterior ACC (pACC) projections, and to evaluate the effects of direct optogenetic manipulation of such projections on mechanical nociception and spontaneous ongoing pain in mice with Spared Nerve Injury (SNI). CTB488 tract-tracing in male PVCrexAi9 mice revealed a small proportion of PV+ projections from the pIC to the pACC. Electrophysiological analysis confirmed the existence of synaptic inputs into the pACC by pIC GABAergic cells. Optogenetic stimulation of these pathways did not change mechanical nociception, but induced conditioned place preference behavior responses. Our results suggest the presence of inhibitory projections between the pIC and the pACC which are able to selectively modulate affective aspects of neuropathic pain.
  • Thumbnail Image
    ItemOpen Access
    Mercury-induced toxicity of rat cortical neurons is mediated through N-Methyl-D-Aspartate receptors
    (BioMed Central Ltd., 2012-09-14) Xu, Fenglian; Farkas, Svetlana; Kortbeek, Simone; Chen, Lina; Zhang, Fangxiong; Zamponi, Gerald W.; Syed, Naweed I.
    Mercury is a well-known neurotoxin implicated in a wide range of neurological or psychiatric disorders including autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, depression, mood disorders and tremor. Mercury-induced neuronal degeneration is thought to invoke glutamate-mediated excitotoxicity, however, the underlying mechanisms remain poorly understood. Here, we examine the effects of various mercury concentrations (including pathological levels present in human plasma or cerebrospinal fluid) on cultured, rat cortical neurons.
  • Thumbnail Image
    ItemOpen Access
    A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain
    (Elsevier, 2011-04-01) Hildebrand, Michael E.; Smith, Paula L.; Bladen, Chris; Eduljee, Cyrus; Xie, Jennifer Yanhua; Chen, Lina; Fee-Maki, Molly; Doering, Clinton J.; Mezeyova, Janette; Zhu, Yongbao; Belardetti, Francesco; Pajouhesh, Hassan; Parker, David B.; Arnerić, Stephen Peter; Parmar, Manjeet; Porreca, Frank; Tringham, Elizabeth W.; Zamponi, Gerald W.; Snutch, Terrance Preston
    Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.
  • Thumbnail Image
    ItemOpen Access
    Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition
    (BioMed Central Ltd., 2010-02-03) Tedford, Hugo William; Kisilevsky, Alexandra E.; Vieira, Luciene Bruno; Varela, Diego L.; Chen, Lina; Zamponi, Gerald W.
    Direct interaction with the beta subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel alpha1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gbeta1gamma2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the alpha1 subunit, residues which are surface-exposed in the presence of the calcium channel beta subunit, contribute significantly to the functional inhibition by Gbeta1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gbeta1gamma2 subunits and with several isoforms of the auxiliary beta subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel.
  • Thumbnail Image
    ItemOpen Access
    Small organic molecule disruptors of Cav3.2 - USP5 interactions reverse inflammatory and neuropathic pain
    (BioMed Central Ltd., 2015-12) Gadotti, Vinícius Maria; Caballero, Agustin Garcia; Berger, N. Daniel; Gladding, Clare M.; Chen, Lina; Pfeifer, Tom A.; Zamponi, Gerald W.
    Cav3.2 channels facilitate nociceptive transmission and are upregulated in DRG neurons in response to nerve injury or peripheral inflammation. We reported that this enhancement of Cav3.2 currents in afferent neurons is mediated by deubiquitination of the channels by the deubiquitinase USP5, and that disrupting USP5/Cav3.2 channel interactions protected from inflammatory and neuropathic pain.
  • Thumbnail Image
    ItemOpen Access
    SUMOylation regulates USP5-Cav3.2 calcium channel interactions
    (2019-08-27) Garcia-Caballero, Agustin; Zhang, Fang-Xiong; Chen, Lina; M’Dahoma, Said; Huang, Junting; Zamponi, Gerald W
    Abstract Cav3.2 calcium channels play a key role in nociceptive signaling in the primary afferent pain pathway. We have previously reported the regulation of Cav3.2 calcium channels by the deubiquitinase USP5 and its importance for regulating peripheral transmission of pain signals. Here we describe the regulation of the Cav3.2-USP5 interaction by SUMOylation. We show that endogenous USP5 protein expressed in dorsal root ganglia undergoes SUMOylation, and the level of USP5 SUMOylation is reduced following peripheral nerve injury. SUMO prediction software identified several putative lysines that have the propensity to be targets for SUMO conjugation. A series of single lysine substitutions in an mCherry tagged USP5 construct followed by expression in tsA-201 cells identified lysine K113 as a key target for USP5 SUMO2/3 modification. Finally, Cav3.2 calcium channel immunoprecipitates revealed a stronger interaction of Cav3.2 with a SUMO2/3 resistant USP5-K113R mutant, indicating that SUMO2/3 modification of USP5 reduces its affinity for the calcium channel Cav3.2. Collectively, our data suggest that dysregulation of USP5 SUMOylation after peripheral nerve injury may contribute to the well described alteration in Cav3.2 channel activity during neuropathic pain states.
  • Thumbnail Image
    ItemOpen Access
    T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B
    (2018-05-02) Garcia-Caballero, Agustin; Zhang, Fang-Xiong; Hodgkinson, Victoria; Huang, Junting; Chen, Lina; Souza, Ivana A; Cain, Stuart; Kass, Jennifer; Alles, Sascha; Snutch, Terrance P; Zamponi, Gerald W
    Abstract This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/β) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/β) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/β) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.
  • Thumbnail Image
    ItemOpen Access
    TNF-α mediated upregulation of NaV1.7 currents in rat dorsal root ganglion neurons is independent of CRMP2 SUMOylation
    (2019-12-30) de Macedo, Flávio H P; Aires, Rosária D; Fonseca, Esdras G; Ferreira, Renata C M; Machado, Daniel P D; Chen, Lina; Zhang, Fang-Xiong; Souza, Ivana A; Lemos, Virgínia S; Romero, Thiago R L; Moutal, Aubin; Khanna, Rajesh; Zamponi, Gerald W; Cruz, Jader S
    Abstract Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.