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Ion Channel Localization pp 1–15Cite as

Fluorescent Calcium Antagonists Tools for Imaging of L-Type Calcium Channels in Living Cells

Tools for Imaging of L-Type Calcium Channels in Living Cells

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Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Different types of voltage-dependent calcium (Ca2+) channels (VCCs) in the plasma-membrane control depolarization-induced Ca2+ entry into cells, thereby serving important physiological functions, including excitationcontraction coupling, neurotransmitter and hormone release, and neuronal plasticity (for review, see refs. 1–4). Their function is fine-tuned by a variety of modulators, such as enzymes and G-proteins (5). In addition the spatial distribution of VCCs over the plasma membrane seems to be of fundamental importance for their contribution to cellular function (6). On the molecular level, VCCs are complexes of a pore-forming α1 subunit, an extracellular α2 subunit attached to the membrane by linkage to the transmembrane ° subunit, and a γ subunit, which is a transmembrane glycoprotein (for review, see ref. 4). At the time of this writing 10 genes encoding α1 subunits are known: α1A–α1I, and α1S (7–9). Four of these (α1C, α1D, α1F, and α1S) encode L-type calcium channels (LTCCs), which are defined by distinct physiological and pharmacological properties, including activation at strong depolarized voltages, slow inactivation, large single channel conductance, and block by Ca2+ antagonists (10,11). A number of chemically unrelated drugs, such as nifedipine (a dihydropyridine, DHP; Fig. 1A), verapamil (a phenylalkylamine, PAA; Fig. 1B), and diltiazem (a benzothiazepine, BTZ) belong to the group of Ca2+ antagonists, which are widely used in the therapy of cardiovascular disorders (for review, see ref. 12). LTCCs are expressed in most neuronal cell types, are the primary type in skeletal muscle cells, and are responsible for the inward movement of calcium ions that initiates contraction of cardiac and smooth muscle cells. The functional role of LTCCs in neurons is still under investigation. Several lines of evidence indicate that LTCCs have a crucial role in regulation of gene transcription by activation of the Ca2+- and cAMP-dependent transcription factor CREB (13).

Representative structures of Ca2+ antagonists and experimental strategy. Prototypic examples of dihydropyridines (A), phenylalklyamines (B), and fluorescent Ca2+ antagonists (C) are shown. (D) During incubation of living cells, fluorescent Ca2+ antagonists bind to LTCCs. After removal of unbound ligands only specific label remains (for possible complications see Section 2.

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References

  1. Miller, R. J. (1988) Calcium signaling in neurons. TINS 11, 415–419.

    PubMed  CAS  Google Scholar 

  2. Kennedy, M. B. (1989) Regulation of neuronal function by calcium. TINS 12, 417–420.

    PubMed  CAS  Google Scholar 

  3. Bliss, T. V. P. and Collingridge, G. L. (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39.

    Article  PubMed  CAS  Google Scholar 

  4. Catterall, W. A. (1998) Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24, 307–323.

    Article  PubMed  CAS  Google Scholar 

  5. Dolphin, A. C. (1998) Mechanisms of modulation of voltage-dependent calcium channels by G proteins. J. Physiol. (Lond.) 506, 3–11.

    Article  CAS  Google Scholar 

  6. Destexhe, A., Contreras, D., Steriade, M., Sejnowski, T. J., and Huguenard, J. R. (1996) In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons. J. Neurosci.1 16, 169–185.

    CAS  Google Scholar 

  7. Birnbauer, L. (1994) The naming of voltage-gated calcium channels. Neuron 13, 505–506.

    Article  Google Scholar 

  8. Perez-Reyes, E., Cribbs, L. L., Daud, A., Lacerda, A. E., Barclay, J., Williamson, M. P., et al. (1998) Molecular characterization of a neuronal lowvoltage-activated T-type calcium channel. Nature 391, 896–900.

    Article  PubMed  CAS  Google Scholar 

  9. Lee, J. H., Daud, A. N., Cribbs, L. L., Lacerda, A. E., Pereverzev, A., Klockner, U., et al. (1999) Cloning and expression of a novel member of the low voltageactivated T-type calcium channel family. J. Neurosci. 19, 1912–1921.

    PubMed  CAS  Google Scholar 

  10. Reuter, H. (1983) Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301, 569–574.

    Article  PubMed  CAS  Google Scholar 

  11. Nowycky, M. C., Fox, A. P., and Tsien, R. W. (1985) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316, 440–443.

    Article  PubMed  CAS  Google Scholar 

  12. Mitterdorfer, J., Grabner, M., Kraus, R. L., Hering, S., Prinz, H., Glossmann, H., and Striessnig, J. (1998) Molecular basis of drug interaction with L-type Ca2++ channels. J Bioenerg. Biomembr. 30, 319–334.

    Article  PubMed  CAS  Google Scholar 

  13. Bading, H., Ginty, D. D., and Greenberg, M. E. (1993) Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260, 181–186.

    Article  PubMed  CAS  Google Scholar 

  14. Hell, J. W., Westenbroek, R. E., Elliott, E. M., and Catterall, W. A. (1994) Differential phosphorylation, localization, and function of distinct alpha 1 subunits of neuronal calcium channels. Two size forms for class B, C, and D alpha 1 subunits with different COOH-termini. Ann. N. Y. Acad. Sci. 747, 282–293.

    Article  PubMed  CAS  Google Scholar 

  15. Yan, Z. and Surmeier, D. J. (1996) Muscarinic (m2/m4) receptors reduce Nand P-type Ca2++ currents in rat neostriatal cholinergic interneurons through a fast, membrane-delimited, G-protein pathway. J. Neurosci. 16, 2592–2604.

    PubMed  CAS  Google Scholar 

  16. Plant, T. D., Schirra, C., Katz, E., Uchitel, O. D., and Konnerth, A. (1998) Single-cell RT-PCR and functional characterization of Ca2+ channels in motoneurons of the rat facial nucleus. J. Neurosci. 18, 9573–9584.

    PubMed  CAS  Google Scholar 

  17. Westenbroek, R. E., Sakurai, T., Elliott, E. M., Hell, J. W., Starr, T. V., Snutch, T. P., and Catterall, W. A. (1995) Immunochemical identification and subcellular distribution of the alpha 1A subunits of brain calcium channels. J. Neurosci. 15, 6403–6418.

    PubMed  CAS  Google Scholar 

  18. Christie, B. R., Eliot, L. S., Ito, K. I., Miyakawa, H., and Johnston, D. (1995) Different Ca2++ channels in Soma and dendrites of hippocampal pyramidal neurons mediate spike-induced Ca2+ influx. J. Neurophysiol. 73, 2553–2557.

    PubMed  CAS  Google Scholar 

  19. Magee, J. C. and Johnston, D. (1995) Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal cells. J. Physiol. (Lond.) 487.1, 67–90.

    Google Scholar 

  20. Markram, H., Helm, P. J., and Sakmann, B. (1995) Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons. J. Physiol. (Lond.) 485.1, 1–20.

    Google Scholar 

  21. Munsch, T., Budde, T., and Pape, H.-C. (1997) Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons. Neuroreport 8, 2411–2418.

    Article  PubMed  CAS  Google Scholar 

  22. Knaus, H. G., Moshammer, T., Friederich, K., Kang, H. C., Haugland, R. P., and Glossmann, H. (1992) In vivo labeling of L-type channels by flourescent dihydropyridines: evidence for a functional, extracellular heparin-binding site. PNAS 89, 3586–3590.

    Article  PubMed  CAS  Google Scholar 

  23. Knaus, H. G., Moshammer, T., Kang, H. C., Haugland, R. P., and Glossmann, H. (1992) A unique fluorescent phenylalkylamine probe for L-type Ca2++ channels. Coupling of phenylalkylamine receptors to Ca2+ and dihydropyridine binding sites. J. Biol. Chem. 267, 2179–2189.

    PubMed  CAS  Google Scholar 

  24. Srinivasan, Y., Guzikowski, A. P., Haugland, R. P., and Angelides, K. J. (1990) Distribution and lateral mobility of glycine receptors on cultured spinal cord neurons. J. Neurosci. 10, 985–995.

    PubMed  CAS  Google Scholar 

  25. Brauns, T., Cai, Z. W., Kimball, S. D., Kang, K. C., Haugland, R. P., Berger, W., et al. (1995) Benzothiazepine binding domain of purified L-type calcium channels: direct labeling using a novel fluorescent diltiazem analogue. Biochemistry 34, 3461–3469.

    Article  PubMed  CAS  Google Scholar 

  26. Schild, H., Geiling, H., and Bischofberger, J. (1995) Imaging of L-type channels in olfactory blub neurons using flourescent dihydropyridine and a styryl dye. J. Neurosci. Methods 59, 183–190.

    Article  PubMed  CAS  Google Scholar 

  27. Budde, T., Munsch, T., and Pape, H.-C. (1998) Distribution of L-type calcium channels in rat thalamic neurons. Eur. J. Neurosci. 10, 586–597.

    Article  PubMed  CAS  Google Scholar 

  28. Ferry, D. R., Goll, A., and Glossmann, H. (1983) Differential labelling of putative skeletal muscle calcium channels by [3H]-nifedipine, [3H]-nitrendipine, [3H]-nimodipine and [3H]-PN 200 110. Naunyn Schmiedebergs Arch. Pharmacol. 323, 276–277.

    Article  PubMed  CAS  Google Scholar 

  29. Rios, E. and Pizarro, G. (1991) Voltage sensor of excitation-contraction coupling in skeletal muscle. Physiol. Rev. 71, 849–908.

    PubMed  CAS  Google Scholar 

  30. Leclerc, C., Duprat, A. M., and Moreau, M. (1995) In vivo labelling of L-type Ca2+ channels by flourescent dihydropyridine: correlation between ontogen esis of the channels and the acquisition of neural competence in ectoderm cells from Pleurodeles waltl embryos. Cell Calcium 17, 216–224.

    Article  PubMed  CAS  Google Scholar 

  31. Bechem, M., Hebisch, S., and Schramm, M. (1988) Ca2+ agonists: new, sensitive probes for Ca2+ channels. TIBS 9, 257–261.

    CAS  Google Scholar 

  32. Robitaille, R., Bourque, M. J., and Vandaele, S. (1996) Localization of L-type Ca2+ channels at perisynaptic glia cells of the frog neuromuscular junction. J. Neurosci. 16, 148–158.

    PubMed  CAS  Google Scholar 

  33. Vallee, N., Briere, C., Petitprez, M., Barthou, H., Souvre, A., and Alibert, G. (1997) Studies on ion channel antagonist-binding sites in sunflower protoplasts. FEBS Lett. 411, 115–118.

    Article  PubMed  CAS  Google Scholar 

  34. Nowycky, M. C., Fox, A. P., and Tsien, R. W. (1985) Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644. PNAS 82, 2178–2182.

    Article  PubMed  CAS  Google Scholar 

  35. Nachman-Clewner, M., St Jules, R., and Townes-Anderson, E. (1999) L-type calcium channels in the photoreceptor ribbon synapse: localization and role in plasticity. J. Comp. Neurol. 415, 1–16.

    Article  PubMed  CAS  Google Scholar 

  36. Puro, D. G., Hwang, J. J., Kwon, O. J., and Chin, H. (1996) Characterization of an L-type calcium channel expressed by human retinal Muller (glial) cells. Mol. Brain. Res. 37, 41–48.

    Article  PubMed  CAS  Google Scholar 

  37. Shitaka, Y., Matsuki, N., Saito, H., and Katsuki, H. (1996) Basic fibroblast growth factor increases functional L-type Ca2+ channels in fetal rat hippocampal neurons: implications for neurite morphogenesis in vitro. J. Neurosci. 16, 6476–6489.

    PubMed  CAS  Google Scholar 

  38. Goligorsky, M. S., Colflesh, D., Gordienko, D., and Moore, L. C. (1995) Branching points of renal resistance arteries are enriched in L-type calcium channels and initiate vasoconstriction. Am. J. Physiol. 268, F251–257.

    PubMed  CAS  Google Scholar 

  39. Poggi, A., Rubartelli, A., and Zocchi, M. R. (1998) Involvement of dihydropyridine-sensitive calcium channels in human dendritic cell function. Competition by HIV-1 Tat. J. Biol. Chem. 273, 7205–7209.

    Article  PubMed  CAS  Google Scholar 

  40. Brauns, T., Prinz, H., Kimball, S. D., Haugland, R. P., Striessnig, J., and Glossmann, H. (1997) L-type calcium channels: binding domains for dihydropyridines and benzothiazepines are located in close proximity to each other. Biochemistry 36, 3625–3631.

    Article  PubMed  CAS  Google Scholar 

  41. Berger, W., Prinz, H., Striessnig, J., Kang, H. C., Haugland, R., and Glossmann, H. (1994) Complex molecular mechanism for dihydropyridine binding to Ltype Ca(2+)-channels as revealed by fluorescence resonance energy transfer. Biochemistry 33, 11,875–11,883.

    Article  PubMed  CAS  Google Scholar 

  42. Tsien, R. W., Hess, P., McCleskey, E. W., and Rosenberg, R. L. (1987) Calcium channels: mechanisms of selectivity, permeation, and block. Annu. Rev. Biophys. Biophys. Chem. 16, 265–290.

    Article  PubMed  CAS  Google Scholar 

  43. Tang, S., Mikala, G., Bahinski, A., Yatani, A., Varadi, G., and Schwartz, A. (1993) Molecular localization of ion selectivity sites within the pore of a human L-type cardiac calcium channel. J. Biol. Chem. 268, 13,026–13,029.

    PubMed  CAS  Google Scholar 

  44. Yang, J., Ellinor, P. T., Sather, W. A., Zang, J. F., and Tsien, R. W. (1993) Molecular determinants of Ca2++ selectivity and ion permeation in L-type Ca2+ channels. Nature 366, 158–161.

    Article  PubMed  CAS  Google Scholar 

  45. Johnson, J. D. (1983) Allosteric interactions among drug binding sites on calmodulin. Biochem. Biophys. Res. Commun. 112, 787–793.

    Article  PubMed  CAS  Google Scholar 

  46. Dunn, S. M. and Bladen, C. (1992) Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in the fluorescence of felodipine. Biochemistry 31, 4039–4045.

    Article  PubMed  CAS  Google Scholar 

  47. Minarovic, I. and Meszaros, L. G. (1998) Fluorescent probing with felodipine of the dihydropyridine receptor and its interaction with the ryanodine receptor calcium release channel. Biochem. Biophys. Res. Commun. 244, 519–524.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Institute of Physiology, Otto-von-Guericke University, Magdeburg, Germany

    Thomas Budde

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  1. Thomas Budde
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Editors and Affiliations

  1. University of Michigan, Ann Arbor, MI

    Anatoli N. Lopatin

  2. Washington University Medical School, St. Louis, MO

    Colin G. Nichols

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© 2001 Human Press Inc., Totowa, NJ

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Budde, T. (2001). Fluorescent Calcium Antagonists Tools for Imaging of L-Type Calcium Channels in Living Cells. In: Lopatin, A.N., Nichols, C.G. (eds) Ion Channel Localization. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-118-3:1

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  • DOI: https://doi.org/10.1385/1-59259-118-3:1

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-833-2

  • Online ISBN: 978-1-59259-118-3

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