1071. Development and Characterization of HSV-1 Vector-Expressed Dominant Negative PKC epsilon for Pain Therapy

Srinivasan, Rahul; Sculptoreanu, Adrian; Wolfe, Darren; Wechuck, James; Watkins, Simon; de Groat, William C.; Glorioso, Joseph C.
June 2006
Molecular Therapy;Jun2006, Vol. 13, pS411
Academic Journal
The vanilloid receptor (TRPV1) is an important pro-nociceptive Ca+2 channel, the inhibition of which attenuates pain. Recent studies have shown that TRPV1 phosphorylation by protein kinase C epsilon (PKCe) causes receptor sensitization and hyperalgesia. Inhibition of PKCe activity in nociceptors can hence be an approach to treat chronic pain. We previously reported replication defective HSV-1 vectors as a means to efficiently deliver analgesic gene products to rat dorsal root ganglion (DRG) neurons in-vivo. In this study, we recombined dominant negative PKCe (DNPKC) deleted for the C2 and C1b domains and the catalytic region into a replication defective HSV-1 vector (called vHDNPKC). DNPKC was fused to a green fluorescent protein (GFP) coding sequence and placed under transcriptional control of the HCMV immediate early promoter. Live cell imaging of vHDNPKC-infected Vero and U2OS cells displayed perinuclear green fluorescence that translocated to the plasma membrane following stimulation with the PKC activator, PMA. Whole-cell electrophysiological recordings of capsaicin-induced TRPV1 currents in vHDNPKC-infected adult rat DRG neurons demonstrated significantly smaller current amplitudes (2.5 fold) that desensitized faster (4 fold) than uninfected or vHG-infected controls. Control capsaicin responses were potentiated by isoform-specific PKCe translocation following stimulation with a specific bradykinin receptor 2 agonist, HYP3-bradykinin. HYP3-bradykinin failed to potentiate capsaicin currents in vHPKCDN-infected neurons, while the non-specific PKC activator PDBu caused a slow drift current that was antagonized by the PKC inhibitor bisindolylmaleimide. This result demonstrates isoform-specific inhibition of PKCe activity. Taken together, the data demonstratean in-vitro inhibition of TRPV1 function in nociceptive neurons by HSV-1 vector-expressed dominant negative PKCe. This vector can therefore be used as a viable approach for the in-vivo induction of analgesia.Molecular Therapy (2006) 13, S411–S411; doi: 10.1016/j.ymthe.2006.08.1170


Related Articles

  • Activation Mechanisms of Protein Kinase C: Maturation, Catalytic Activation, and Targeting. Shirai, Yasuhito; Saito, Naoaki // Journal of Biochemistry;2002, Vol. 132 Issue 5, p663 

    The biological function of protein kinase C (PKC) depends on its catalytic activity and spatial localization. Its catalytic competence and localization in the resting state are regulated by serine/threonine phosphorylations, i.e., “maturation.” Upon stimulation of various receptors,...

  • Lymphoid signal transduction mechanisms linked to cellular prion protein. Mazzoni, I. E.; Ledebur Jr., H. C.; Paramithiotis, E.; Cashman, N. // Biochemistry & Cell Biology;Oct2005, Vol. 83 Issue 5, p644 

    The normal cellular isoform of the prion protein (PrPC) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed widely, including in lymphoid cells. We compared lectin-induced mitogenesis and selected cell signaling pathways in splenocytes from wild-type BALB/c mice and...

  • The linoleic acid derivative DCP-LA increases membrane surface localization of the α7 ACh receptor in a protein 4.1N-dependent manner. KANNO, Takeshi; TSUCHIYA, Ayako; TANAKA, Akito; NISHIZAKI, Tomoyuki // Biochemical Journal;3/1/2013, Vol. 450 Issue 2, p303 

    In yeast two-hybrid screening, protein 4.1N, a scaffolding protein, was identified as a binding partner of the α7 ACh (acetylcholine) receptor. For rat hippocampal slices, the linoleic acid derivative DCP-LA {8-[2-(2-pentyl-cyclopropylmethyl)- cyclopropyl]-octanoic acid} increased the...

  • NMDA receptor activity downregulates KCC2 resulting in depolarizing GABAA receptor-mediated currents. Lee, Henry H. C.; Deeb, Tarek Z.; Walker, Joshua A.; Davies, Paul A.; Moss, Stephen J. // Nature Neuroscience;Jun2011, Vol. 14 Issue 6, p736 

    KCC2 is a neuron-specific K+-Cl− co-transporter that maintains a low intracellular Cl− concentration that is essential for hyperpolarizing inhibition mediated by GABAA receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release....

  • An aPKC-Exocyst Complex Controls Paxillin Phosphorylation and Migration through Localised JNK1 Activation. Rosse, Carine; Formstecher, Etienne; Boeckeler, Katrina; Yingming Zhao; Kremerskothen, Joachim; White, Michael D.; Camonis, Jacques H.; Parker, Peter J. // PLoS Biology;Nov2009, Vol. 7 Issue 11, p1 

    Atypical protein kinase C (aPKC) isoforms have been implicated in cell polarisation and migration through association with Cdc42 and Par6. In distinct migratory models, the Exocyst complex has been shown to be involved in secretory events and migration. By RNA interference (RNAi) we show that...

  • Biochemical and Cell Biological Mechanisms of Cholecystokinin Receptor Regulation. Laurence J. Miller; Maoqing Dong; Kaleeckal G. Harikumar; Cayle S. Lisenbee // Current Topics in Medicinal Chemistry;Jun2007, Vol. 7 Issue 12, p1166 

    Regulation of the cholecystokinin receptor is accomplished by biochemical and cell biological mechanisms. The major mechanism for biochemical regulation involves phosphorylation of serine and threonine residues within the receptor's intracellular third loop and carboxyl-terminal tail. This form...

  • aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb. Smith, Christian A.; Lau, Kimberly M.; Rahmani, Zohra; Dho, Sascha E.; Brothers, Greg; Ye Min She; Berry, Donna M.; Bonneil, Eric; Thibault, Pierre; Schweisguth, Fran�ois; Le Borgne, Roland; McGlade, C. Jane // EMBO Journal;1/24/2007, Vol. 26 Issue 2, p468 

    In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been...

  • Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C. Specht, Christian G; Grünewald, Nora; Pascual, Olivier; Rostgaard, Nina; Schwarz, Günter; Triller, Antoine // EMBO Journal;9/21/2011, Vol. 30 Issue 18, p3842 

    Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the ?-subunit of the GlyR with the synaptic scaffold protein gephyrin. An...

  • Thapsigargin-Induced Calcium Entry and Apoptotic Death of Neutrophils Are Blocked by Activation of Protein Kinase C. Lucas, Miguel; Díaz, Pilar // Pharmacology;Sep2001, Vol. 63 Issue 3, p191 

    Intracellular free calcium ([Ca[sup 2+] ][sub i] ) concentration, free oxygen radical (FOR) production, DNA breakdown, and plasma membrane phosphorylation were studied in human neutrophils activated with thapsigargin and phorbol myrisate acetate (PMA). Thapsigargin produced a rapid and sustained...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics