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ORL1 receptor–mediated internalization of N-type calcium channels

Abstract

The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged (30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with μ-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.

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Figure 1: Visualization of ORL1 receptor and N-type channel internalization in response to ORL1 receptor activation.
Figure 2: Co-internalization of CFP-Cav2.2 and ORL1-YFP receptors.
Figure 3: Specificity of the receptor-mediated internalization of Cav2.2.
Figure 4: Electrophysiological and biochemical evidence of Cav2.2 internalization.
Figure 5: Internalization of Cav2.2 channels in a subpopulation of acutely dissociated DRG neurons.
Figure 6: Cav2.2 internalization decreases calcium influx into cultured DRG neurons.

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References

  1. Saegusa, H. et al. Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel. EMBO J. 20, 2349–2356 (2001).

    Article  CAS  Google Scholar 

  2. Scott, D.A., Wright, C.E. & Angus, J.A. Actions of intrathecal omega-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat. Eur. J. Pharmacol. 451, 279–286 (2002).

    Article  CAS  Google Scholar 

  3. Staats, P.S. et al. Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. J. Am. Med. Assoc. 291, 63–70 (2004).

    Article  CAS  Google Scholar 

  4. Toth, P.T., Shekter, L.R., Ma, G.H., Philipson, L.H. & Miller, R.J. Selective G-protein regulation of neuronal calcium channels. J. Neurosci. 16, 4617–4624 (1996).

    Article  CAS  Google Scholar 

  5. Bourinet, E., Soong, T.W., Stea, A. & Snutch, T.P. Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc. Natl. Acad. Sci. USA 93, 1486–1491 (1996).

    Article  CAS  Google Scholar 

  6. Connor, M. & Christie, M.J. Modulation of Ca2+ channel currents of acutely dissociated rat periaqueductal grey neurons. J. Physiol. (Lond.) 509, 47–58 (1998).

    Article  CAS  Google Scholar 

  7. Borgland, S.L., Connor, M. & Christie, M.J. Nociceptin inhibits calcium channel currents in a subpopulation of small nociceptive trigeminal ganglion neurons in mouse. J. Physiol. (Lond.) 536, 35–47 (2001).

    Article  CAS  Google Scholar 

  8. Cooper, C.B. et al. Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein beta subunit isoform. J. Biol. Chem. 275, 40777–40781 (2000).

    Article  CAS  Google Scholar 

  9. von Zastrow, M., Svingos, A., Haberstock-Debic, H. & Evans, C. Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs. Curr. Opin. Neurobiol. 13, 348–353 (2003).

    Article  CAS  Google Scholar 

  10. Bunzow, J.R. et al. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type. FEBS Lett. 347, 284–288 (1994).

    Article  CAS  Google Scholar 

  11. Wick, M.J. et al. Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors. Brain Res. Mol. Brain Res. 27, 37–44 (1994).

    Article  CAS  Google Scholar 

  12. Mollereau, C. et al. ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett. 341, 33–38 (1994).

    Article  CAS  Google Scholar 

  13. Reinscheid, R.K. et al. Orphanin FQ: a neuropeptide that activates an opioid-like G protein-coupled receptor. Science 270, 792–794 (1995).

    Article  CAS  Google Scholar 

  14. Knoflach, F., Reinscheid, R.K., Civelli, O. & Kemp, J.A. Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J. Neurosci. 16, 6657–6664 (1996).

    Article  CAS  Google Scholar 

  15. Larsson, K.P., Olsen, U.B. & Hansen, A.J. Nociceptin is a potent inhibitor of N-type Ca2+ channels in rat sympathetic ganglion neurons. Neurosci. Lett. 296, 121–124 (2000).

    Article  CAS  Google Scholar 

  16. Neal, C.R., Jr. et al. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J. Comp. Neurol. 406, 503–547 (1999).

    Article  CAS  Google Scholar 

  17. Yamamoto, T., Nozaki-Taguchi, N. & Kimura, S. Effects of intrathecally administered nociceptin, an opioid receptor-like1 (ORL1) receptor agonist, on the thermal hyperalgesia induced by unilateral constriction injury to the sciatic nerve in the rat. Neurosci. Lett. 224, 107–110 (1997).

    Article  CAS  Google Scholar 

  18. Yamamoto, T., Nozaki-Taguchi, N. & Kimura, S. Analgesic effect of intrathecally administered nociceptin, an opioid receptor-like1 receptor agonist, in the rat formalin test. Neuroscience 81, 249–254 (1997).

    Article  CAS  Google Scholar 

  19. Abdulla, F.A. & Smith, P.A. Nociceptin inhibits T-type Ca2+ channel current in rat sensory neurons by a G-protein-independent mechanism. J. Neurosci. 17, 8721–8728 (1997).

    Article  CAS  Google Scholar 

  20. Beedle, A.M. et al. Agonist-independent modulation of N-type calcium channels by ORL1 receptors. Nat. Neurosci. 7, 118–125 (2004).

    Article  CAS  Google Scholar 

  21. Andoh, T., Itoh, M. & Kuraishi, Y. Nociceptin gene expression in rat dorsal root ganglia induced by peripheral inflammation. Neuroreport 8, 2793–2796 (1997).

    Article  CAS  Google Scholar 

  22. Jia, Y., Linden, D.R., Serie, J.R. & Seybold, V.S. Nociceptin/orphanin FQ binding increases in superficial laminae of the rat spinal cord during persistent peripheral inflammation. Neurosci. Lett. 250, 21–24 (1998).

    Article  CAS  Google Scholar 

  23. Briscini, L., Corradini, L., Ongini, E. & Bertorelli, R. Up-regulation of ORL-1 receptors in spinal tissue of allodynic rats after sciatic nerve injury. Eur. J. Pharmacol. 447, 59–65 (2002).

    Article  CAS  Google Scholar 

  24. Gouarderes, C., Tafani, J.A., Meunier, J.C., Jhamandas, K. & Zajac, J.M. Nociceptin receptors in the rat spinal cord during morphine tolerance. Brain Res. 838, 85–94 (1999).

    Article  CAS  Google Scholar 

  25. Ueda, H., Inoue, M., Takeshima, H. & Iwasawa, Y. Enhanced spinal nociceptin receptor expression develops morphine tolerance and dependence. J. Neurosci. 20, 7640–7647 (2000).

    Article  CAS  Google Scholar 

  26. Spampinato, S., Di Toro, R. & Qasem, A.R. Nociceptin-induced internalization of the ORL1 receptor in human neuroblastoma cells. Neuroreport 12, 3159–3163 (2001).

    Article  CAS  Google Scholar 

  27. Spampinato, S., Di Toro, R., Alessandri, M. & Murari, G. Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells. Cell. Mol. Life Sci. 59, 2172–2183 (2002).

    Article  CAS  Google Scholar 

  28. Corbani, M., Gonindard, C. & Meunier, J.C. Ligand-regulated internalization of the opioid receptor-like 1: a confocal study. Endocrinology 145, 2876–2885 (2004).

    Article  CAS  Google Scholar 

  29. Mandyam, C.D., Thakker, D.R., Christensen, J.L. & Standifer, K.M. Orphanin FQ/nociceptin-mediated desensitization of opioid receptor-like 1 receptor and mu opioid receptors involves protein kinase C: a molecular mechanism for heterologous cross-talk. J. Pharmacol. Exp. Ther. 302, 502–509 (2002).

    Article  CAS  Google Scholar 

  30. Dale, L.B., Seachrist, J.L., Babwah, A.V. & Ferguson, S.S. Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases. J. Biol. Chem. 279, 13110–13118 (2004).

    Article  CAS  Google Scholar 

  31. Becker, J.A. et al. Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library. J. Biol. Chem. 274, 27513–27522 (1999).

    Article  CAS  Google Scholar 

  32. Mills, R.H., Sohn, R.K. & Micevych, P.E. Estrogen-induced mu-opioid receptor internalization in the medial preoptic nucleus is mediated via neuropeptide Y-Y1 receptor activation in the arcuate nucleus of female rats. J. Neurosci. 24, 947–955 (2004).

    Article  CAS  Google Scholar 

  33. Abdulla, F.A. & Smith, P.A. Axotomy reduces the effect of analgesic opioids yet increases the effect of nociceptin on dorsal root ganglion neurons. J. Neurosci. 18, 9685–9694 (1998).

    Article  CAS  Google Scholar 

  34. Sutton, K.G., Martin, D.J., Pinnock, R.D., Lee, K. & Scott, R.H. Gabapentin inhibits high-threshold calcium channel currents in cultured rat dorsal root ganglion neurones. Br. J. Pharmacol. 135, 257–265 (2002).

    Article  CAS  Google Scholar 

  35. Scott, R.H. et al. Inhibition of neuronal high voltage-activated calcium channels by insect peptides: a comparison with the actions of omega-conotoxin GVIA. Neuropharmacology 36, 195–208 (1997).

    Article  CAS  Google Scholar 

  36. Dolphin, A.C. G protein modulation of voltage-gated calcium channels. Pharmacol. Rev. 55, 607–627 (2003).

    Article  CAS  Google Scholar 

  37. Zamponi, G.W. Determinants of G protein inhibition of presynaptic calcium channels. Cell Biochem. Biophys. 34, 79–94 (2001).

    Article  CAS  Google Scholar 

  38. Zamponi, G.W., Bourinet, E., Nelson, D., Nargeot, J. & Snutch, T.P. Crosstalk between G proteins and protein kinase C mediated by the calcium channel α1 subunit. Nature 385, 442–446 (1997).

    Article  CAS  Google Scholar 

  39. De Waard, M. et al. Direct binding of G-protein βγcomplex to voltage-dependent calcium channels. Nature 385, 446–450 (1997).

    Article  CAS  Google Scholar 

  40. Herlitze, S. et al. Modulation of Ca2+ channels by G-protein βγ subunits. Nature 380, 258–262 (1996).

    Article  CAS  Google Scholar 

  41. Ikeda, S.R. Voltage-dependent modulation of N-type calcium channels by G-protein βγ subunits. Nature 380, 255–258 (1996).

    Article  CAS  Google Scholar 

  42. Bean, B.P. Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340, 153–156 (1989).

    Article  CAS  Google Scholar 

  43. Patil, P.G. et al. Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels. Biophys. J. 71, 2509–2521 (1996).

    Article  CAS  Google Scholar 

  44. Hao, J.X., Wiesenfeld-Hallin, Z. & Xu, X.J. Lack of cross-tolerance between the antinociceptive effect of intrathecal orphanin FQ and morphine in the rat. Neurosci. Lett. 223, 49–52 (1997).

    Article  CAS  Google Scholar 

  45. Hatakeyama, S. et al. Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca2+ channels. Neuroreport 12, 2423–2427 (2001).

    Article  CAS  Google Scholar 

  46. Lutfy, K. et al. Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors. J. Neurosci. 23, 10331–10337 (2003).

    Article  CAS  Google Scholar 

  47. Jhamandas, K.H., Sutak, M. & Henderson, G. Antinociceptive and morphine modulatory actions of spinal orphanin FQ. Can. J. Physiol. Pharmacol. 76, 314–324 (1998).

    Article  CAS  Google Scholar 

  48. Zaratin, P.F. et al. Modification of nociception and morphine tolerance by the selective opiate receptor-like orphan receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). J. Pharmacol. Exp. Ther. 308, 454–461 (2004).

    Article  CAS  Google Scholar 

  49. Altier, C. et al. Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79. J. Biol. Chem. 277, 33598–33603 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Snutch for providing wild-type calcium channel subunits. We thank S. Jarvis for help with HA-Cav2.2 cDNA, and are grateful to J. Hamid, C. Barrere and J. Nargeot for assistance and technical discussions. This work was supported by operating grants to G.W.Z. and S.S.G.F. from the Canadian Institutes of Health Research (CIHR), and grants to E.B. from the Institut UPSA de la Douleur and the Fondation Paul Hamel, and by a CNRS International Programs for Scientific Cooperation (PICS) exchange program. G.W.Z. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research (AHFMR) and a Canada Research Chair in Molecular Neurobiology. S.S.G.F. is also a Canada Research Chair. C.A. holds postdoctoral fellowships from the AHFMR and the Heart and Stroke Foundation of Canada, R.M.E. holds a postdoctoral fellowship from the AHFMR, H.K. holds a Canada Graduate Scholarship and an AHFMR studentship, J.B.P. holds a Foundation for Fighting Blindness studentship, and B.A.V. holds a CIHR Master's studentship.

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Correspondence to Gerald W Zamponi.

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Supplementary information

Supplementary Fig. 1

Control experiments examining cell type, sizes of nuclei, and distribution of HA-tagged calcium channels. (PDF 430 kb)

Supplementary Fig. 2

Specificity of the ORL1 receptor antibody used in our study. (PDF 327 kb)

Supplementary Methods (PDF 187 kb)

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Altier, C., Khosravani, H., Evans, R. et al. ORL1 receptor–mediated internalization of N-type calcium channels. Nat Neurosci 9, 31–40 (2006). https://doi.org/10.1038/nn1605

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