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Genetically encoded molecules for inducibly inactivating CaV channels

Abstract

Voltage-gated Ca2+ (CaV) channels are central to the biology of excitable cells, and therefore regulating their activity has widespread applications. We describe genetically encoded molecules for inducibly inhibiting CaV channels (GEMIICCs). GEMIICCs are derivatives of Rem, a Ras-like GTPase that constitutively inhibits Ca2+ currents (ICa). C terminus–truncated Rem1–265 lost the ability to inhibit ICa owing to loss of membrane targeting. Fusing the C1 domain of protein kinase Cγ to yellow fluorescent protein (YFP)-Rem1–265 generated a molecule that rapidly translocated from cytosol to plasma membrane with phorbol-12,13-dibutyrate in human embryonic kidney cells. Recombinant CaV2.2 and CaV1.2 channels were inhibited concomitantly with C1PKCγ-YFP-Rem1–265 membrane translocation. The generality of the approach was confirmed by creating a GEMIICC using rapamycin-dependent heterodimerization of YFP-FKBP-Rem1–265 and a constitutively membrane-targeted rapamycin-binding domain. GEMIICCs reduced ICa without diminishing gating charge, thereby ruling out decreased number of surface channels and voltage-sensor immobilization as mechanisms for inhibition. We introduce small-molecule–regulated GEMIICCs as potent tools for rapidly manipulating Ca2+ signals in excitable cells.

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Figure 1: Role of membrane targeting in Rem-mediated inhibition of CaV2.2 channels.
Figure 2: Basal effects of PdBu on CaV2.2 channel currents.
Figure 3: Generation of a GEMIICC using PdBu-induced membrane translocation of C1PKCγ-YFP-Rem1–265.
Figure 4: C1PKCγ-YFP-Rem1–265 is a selective GEMIICC.
Figure 5: Generation of a GEMIICC using a rapamycin-mediated heterodimerization strategy.
Figure 6: C1PKCγ-YFP-Rem1–265 GEMIICC inhibits whole-cell ICa without affecting gating current.
Figure 7: Cytosolic- and membrane-targeted Rem proteins have a similarly low affinity for CaVβ3.

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References

  1. Catterall, W.A. Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 16, 521–555 (2000).

    Article  CAS  Google Scholar 

  2. Leone, M. et al. Verapamil in the prophylaxis of episodic cluster headache: a double-blind study versus placebo. Neurology 54, 1382–1385 (2000).

    Article  CAS  Google Scholar 

  3. Kochegarov, A.A. Pharmacological modulators of voltage-gated calcium channels and their therapeutical application. Cell Calcium 33, 145–162 (2003).

    Article  CAS  Google Scholar 

  4. Triggle, D.J. Drug targets in the voltage-gated calcium channel family: why some are and some are not. Assay Drug Dev. Technol. 1, 719–733 (2003).

    Article  CAS  Google Scholar 

  5. Valentino, K. et al. A selective N-type calcium channel antagonist protects against neuronal loss after global cerebral ischemia. Proc. Natl. Acad. Sci. USA 90, 7894–7897 (1993).

    Article  CAS  Google Scholar 

  6. Marek, K.W. & Davis, G.W. Controlling the active properties of excitable cells. Curr. Opin. Neurobiol. 13, 607–611 (2003).

    Article  CAS  Google Scholar 

  7. Karpova, A.Y., Tervo, D.G., Gray, N.W. & Svoboda, K. Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons. Neuron 48, 727–735 (2005).

    Article  CAS  Google Scholar 

  8. George, M.S. & Pitt, G.S. The real estate of cardiac signaling: location, location, location. Proc. Natl. Acad. Sci. USA 103, 7535–7536 (2006).

    Article  CAS  Google Scholar 

  9. Berkefeld, H. et al. BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling. Science 314, 615–620 (2006).

    Article  CAS  Google Scholar 

  10. Muller, A., Kukley, M., Uebachs, M., Beck, H. & Dietrich, D. Nanodomains of single Ca2+ channels contribute to action potential repolarization in cortical neurons. J. Neurosci. 27, 483–495 (2007).

    Article  Google Scholar 

  11. Balijepalli, R.C., Foell, J.D., Hall, D.D., Hell, J.W. & Kamp, T.J. Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for β2-adrenergic regulation. Proc. Natl. Acad. Sci. USA 103, 7500–7505 (2006).

    Article  CAS  Google Scholar 

  12. Finlin, B.S., Crump, S.M., Satin, J. & Andres, D.A. Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases. Proc. Natl. Acad. Sci. USA 100, 14469–14474 (2003).

    Article  CAS  Google Scholar 

  13. Beguin, P. et al. Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature 411, 701–706 (2001).

    Article  CAS  Google Scholar 

  14. Seu, L. & Pitt, G.S. Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases. J. Gen. Physiol. 128, 605–613 (2006).

    Article  CAS  Google Scholar 

  15. Lin, Y., McDonough, S.I. & Lipscombe, D. Alternative splicing in the voltage-sensing region of N-Type CaV2.2 channels modulates channel kinetics. J. Neurophysiol. 92, 2820–2830 (2004).

    Article  CAS  Google Scholar 

  16. Maguire, J. et al. Gem: an induced, immediate early protein belonging to the Ras family. Science 265, 241–244 (1994).

    Article  CAS  Google Scholar 

  17. Finlin, B.S. & Andres, D.A. Rem is a new member of the Rad- and Gem/Kir Ras-related GTP-binding protein family repressed by lipopolysaccharide stimulation. J. Biol. Chem. 272, 21982–21988 (1997).

    Article  CAS  Google Scholar 

  18. Reynet, C. & Kahn, C.R. Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans. Science 262, 1441–1444 (1993).

    Article  CAS  Google Scholar 

  19. Heo, W.D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006).

    Article  CAS  Google Scholar 

  20. Colicelli, J. Human RAS superfamily proteins and related GTPases. Sci. STKE 2004, RE13 (2004).

    PubMed  PubMed Central  Google Scholar 

  21. Arni, S., Keilbaugh, S.A., Ostermeyer, A.G. & Brown, D.A. Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues. J. Biol. Chem. 273, 28478–28485 (1998).

    Article  CAS  Google Scholar 

  22. Chen, H., Puhl, H.L., III, Niu, S.L., Mitchell, D.C. & Ikeda, S.R. Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density. J. Neurosci. 25, 9762–9772 (2005).

    Article  CAS  Google Scholar 

  23. Oancea, E., Teruel, M.N., Quest, A.F. & Meyer, T. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells. J. Cell Biol. 140, 485–498 (1998).

    Article  CAS  Google Scholar 

  24. Yang, J. & Tsien, R.W. Enhancement of N- and L-type calcium channel currents by protein kinase C in frog sympathetic neurons. Neuron 10, 127–136 (1993).

    Article  CAS  Google Scholar 

  25. Maeno-Hikichi, Y. et al. A PKC epsilon-ENH-channel complex specifically modulates N-type Ca2+ channels. Nat. Neurosci. 6, 468–475 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. 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 

  28. Raingo, J., Castiglioni, A.J. & Lipscombe, D. Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors. Nat. Neurosci. 10, 285–292 (2007).

    Article  CAS  Google Scholar 

  29. Finlin, B.S. et al. Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. 280, 41864–41871 (2005).

    Article  CAS  Google Scholar 

  30. Crabtree, G.R. & Schreiber, S.L. Three-part inventions: intracellular signaling and induced proximity. Trends Biochem. Sci. 21, 418–422 (1996).

    Article  CAS  Google Scholar 

  31. Inoue, T., Heo, W.D., Grimley, J.S., Wandless, T.J. & Meyer, T. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat. Methods 2, 415–418 (2005).

    Article  CAS  Google Scholar 

  32. Clackson, T. et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. USA 95, 10437–10442 (1998).

    Article  CAS  Google Scholar 

  33. Beguin, P. et al. Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14–3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation. J. Mol. Biol. 355, 34–46 (2006).

    Article  CAS  Google Scholar 

  34. Sasaki, T. et al. Direct inhibition of the interaction between alpha-interaction domain and beta-interaction domain of voltage-dependent Ca2+ channels by Gem. J. Biol. Chem. 280, 9308–9312 (2005).

    Article  CAS  Google Scholar 

  35. Murata, M., Cingolani, E., McDonald, A.D., Donahue, J.K. & Marban, E. Creation of a genetic calcium channel blocker by targeted gem gene transfer in the heart. Circ. Res. 95, 398–405 (2004).

    Article  CAS  Google Scholar 

  36. Erickson, M.G., Alseikhan, B.A., Peterson, B.Z. & Yue, D.T. Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells. Neuron 31, 973–985 (2001).

    Article  CAS  Google Scholar 

  37. Erickson, M.G., Liang, H., Mori, M.X. & Yue, D.T. FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation. Neuron 39, 97–107 (2003).

    Article  CAS  Google Scholar 

  38. Slimko, E.M., McKinney, S., Anderson, D.J., Davidson, N. & Lester, H.A. Selective electrical silencing of mammalian neurons in vitro by the use of invertebrate ligand-gated chloride channels. J. Neurosci. 22, 7373–7379 (2002).

    Article  CAS  Google Scholar 

  39. White, B., Osterwalder, T. & Keshishian, H. Molecular genetic approaches to the targeted suppression of neuronal activity. Curr. Biol. 11, R1041–R1053 (2001).

    Article  CAS  Google Scholar 

  40. White, B.H. et al. Targeted attenuation of electrical activity in Drosophila using a genetically modified K+ channel. Neuron 31, 699–711 (2001).

    Article  CAS  Google Scholar 

  41. Johns, D.C., Marx, R., Mains, R.E., O'Rourke, B. & Marban, E. Inducible genetic suppression of neuronal excitability. J. Neurosci. 19, 1691–1697 (1999).

    Article  CAS  Google Scholar 

  42. Herlitze, S. & Landmesser, L.T. New optical tools for controlling neuronal activity. Curr. Opin. Neurobiol. 17, 87–94 (2007).

    Article  CAS  Google Scholar 

  43. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

    Article  CAS  Google Scholar 

  44. Zemelman, B.V., Lee, G.A., Ng, M. & Miesenbock, G. Selective photostimulation of genetically chARGed neurons. Neuron 33, 15–22 (2002).

    Article  CAS  Google Scholar 

  45. Zemelman, B.V., Nesnas, N., Lee, G.A. & Miesenbock, G. Photochemical gating of heterologous ion channels: remote control over genetically designated populations of neurons. Proc. Natl. Acad. Sci. USA 100, 1352–1357 (2003).

    Article  CAS  Google Scholar 

  46. Borst, J.G. & Sakmann, B. Calcium influx and transmitter release in a fast CNS synapse. Nature 383, 431–434 (1996).

    Article  CAS  Google Scholar 

  47. Dodge, F.A., Jr. & Rahamimoff, R. Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J. Physiol. (Lond.) 193, 419–432 (1967).

    Article  CAS  Google Scholar 

  48. Wu, L.G. & Saggau, P. Presynaptic calcium is increased during normal synaptic transmission and paired-pulse facilitation, but not in long-term potentiation in area CA1 of hippocampus. J. Neurosci. 14, 645–654 (1994).

    Article  CAS  Google Scholar 

  49. Nattel, S. New ideas about atrial fibrillation 50 years on. Nature 415, 219–226 (2002).

    Article  CAS  Google Scholar 

  50. Hawke, T.J., Kanatous, S.B., Martin, C.M., Goetsch, S.C. & Garry, D.J. Rad is temporally regulated within myogenic progenitor cells during skeletal muscle regeneration. Am. J. Physiol. Cell Physiol. 290, C379–C387 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank W. Wang for technical assistance; D. Andres (University of Kentucky) for HA-tagged Rem constructs; M. Tadross (Johns Hopkins University) for the mem-YFP construct; T. Meyer and T. Inoue (Stanford University) for FRB and FKBP constructs; D. Lipscombe (Brown University) for tsA201 cells stably expressing CaV2.2 channels; and I. Dick for comments on the manuscript. This work was supported by grants from the US National Institutes of Health (to H.M.C.).

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T.Y. designed and performed experiments and analyzed results; Y.S. designed and performed experiments and analyzed results; S.D. performed experiments and analyzed results; T.K. performed experiments; H.M.C. designed and performed experiments, analyzed results and wrote the paper.

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Correspondence to Henry M Colecraft.

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Yang, T., Suhail, Y., Dalton, S. et al. Genetically encoded molecules for inducibly inactivating CaV channels. Nat Chem Biol 3, 795–804 (2007). https://doi.org/10.1038/nchembio.2007.42

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