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
  • Thomas Budde
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


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, 2, 3, 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, 8, 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).
Fig. 1.

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.


BAYK 8644 Thalamic Neuron Neuronal Cell Type Ribbon Synapse Frog Neuromuscular Junction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Miller, R. J. (1988) Calcium signaling in neurons. TINS 11, 415–419.PubMedGoogle Scholar
  2. 2.
    Kennedy, M. B. (1989) Regulation of neuronal function by calcium. TINS 12, 417–420.PubMedGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 4.
    Catterall, W. A. (1998) Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24, 307–323.PubMedCrossRefGoogle Scholar
  5. 5.
    Dolphin, A. C. (1998) Mechanisms of modulation of voltage-dependent calcium channels by G proteins. J. Physiol. (Lond.) 506, 3–11.CrossRefGoogle Scholar
  6. 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.Google Scholar
  7. 7.
    Birnbauer, L. (1994) The naming of voltage-gated calcium channels. Neuron 13, 505–506.CrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.PubMedGoogle Scholar
  10. 10.
    Reuter, H. (1983) Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301, 569–574.PubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedGoogle Scholar
  16. 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.PubMedGoogle Scholar
  17. 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.PubMedGoogle Scholar
  18. 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.PubMedGoogle Scholar
  19. 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. 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. 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.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 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.PubMedGoogle Scholar
  24. 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.PubMedGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 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.PubMedCrossRefGoogle Scholar
  29. 29.
    Rios, E. and Pizarro, G. (1991) Voltage sensor of excitation-contraction coupling in skeletal muscle. Physiol. Rev. 71, 849–908.PubMedGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 31.
    Bechem, M., Hebisch, S., and Schramm, M. (1988) Ca2+ agonists: new, sensitive probes for Ca2+ channels. TIBS 9, 257–261.Google Scholar
  32. 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.PubMedGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 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.PubMedCrossRefGoogle Scholar
  35. 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.PubMedCrossRefGoogle Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. 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.PubMedGoogle Scholar
  38. 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.PubMedGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar
  43. 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.PubMedGoogle Scholar
  44. 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.PubMedCrossRefGoogle Scholar
  45. 45.
    Johnson, J. D. (1983) Allosteric interactions among drug binding sites on calmodulin. Biochem. Biophys. Res. Commun. 112, 787–793.PubMedCrossRefGoogle Scholar
  46. 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.PubMedCrossRefGoogle Scholar
  47. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Human Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Thomas Budde
    • 1
  1. 1.Institute of PhysiologyOtto-von-Guericke UniversityMagdeburgGermany

Personalised recommendations