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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
Protocol
Part of the Methods in Pharmacology and Toxicology book series (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, 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.

Keywords

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.

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Copyright information

© Human Press Inc., Totowa, NJ 2001

Authors and Affiliations

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

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