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Characterization of Calcium Channel Binding

Wei Zheng1

1Merck and Company, North Wales, Pennsylvania

Publication Name: 
Current Protocols in Pharmacology
Unit Number: 
Unit 1.25
DOI: 
10.1002/0471141755.ph0125s14
Online Posting Date: 
November, 2001
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Abstract

Voltage-dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain. Saturation binding of a radioligand to the calcium channel is commonly used to characterize the expression level of the channel protein. Compound competition binding assay is a conventional screening method to determine the affinity of unlabeled compounds for the channel protein. This unit provides detailed experimental methods for two types of radioligand binding assays using [3H]PN200-100 and [125I](conotoxin MVIIA. Voltage-dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Measurement of [3H]PN200-100 Saturation Binding to L-type Calcium Channels in Cardiac Membranes
  • Basic Protocol 2: Measurement of [3H]PN200-100 Displacement Binding to L-type Calcium Channels in Cardiac Membranes
  • Basic Protocol 3: Measurement of [125I]-Conotoxin-MVIIA Saturation Binding to N-type Calcium Channels in Rat Brain Membranes
  • Basic Protocol 4: Measurement of Compound and [125I]-Conotoxin MVIIA Competition Binding to N-type Calcium Channels in Rat Brain Membranes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of [3H]PN200-100 Saturation Binding to L-type Calcium Channels in Cardiac Membranes

 Materials
  • Mouse or rat heart tissue (fresh or previously frozen)
  • L-type binding buffer: 50 mM Tris×Cl, pH 7.2 at room temperature, then chilled to be ice cold
  • [3H]PN200-100 (NEN Life Sciences)
  • 10 µM nitrendipine (for defining nonspecific binding) (ICN or Sigma)
  • L-type wash buffer: 5 mM Tris×Cl, pH 7.2 at room temperature, then chilled to be ice cold
  • Test compounds
  • Dissecting scissors
  • 50-ml centrifuge tubes
  • Polytron homogenizer (Brinkman)
  • Glass homogenizer with Teflon pestle and Tri-R Stir-R variable speed electric motor (Tri-R Instruments)
  • Cheesecloth (e.g., Grade 50; VWR Scientific)
  • 5-ml polypropylene test tubes
  • Glass fiber filters (GF/B type, Whatman)
  • Cell harvester (Brandel Instrument)
  • Scintillation vials
  • Scintillation fluid
  • -scintillation counter
  • Curve fitting programs (e.g., KELL, Biosoft; or Prism, GraphPad Software)

Basic Protocol 3: Measurement of [125I]-Conotoxin-MVIIA Saturation Binding to N-type Calcium Channels in Rat Brain Membranes

 Materials
  • Whole rat brain (fresh or previously frozen)
  • N-type binding buffer: 50 mM Tris×Cl with 0.1% BSA, pH 7.2 at room temperature, then chilled to be ice cold
  • [125I]-conotoxin-MVIIA (NEN Life Sciences)
  • -conotoxin-MVIIA (for defining nonspecific binding; Peninsula Laboratories)
  • Test compounds
  • 0.5% (w/v) polyethyleneimine (PEI)
  • N-type wash buffer: 5 mM Tris×Cl and 150 mM NaCl, pH 7.2 at room temperature, then chilled to be ice cold
  • 50-ml centrifuge tube
  • Glass homogenizer with Teflon pestle and Tri-R Stir-R variable speed electric motor (Tri-R Instruments)
  • 5-ml polypropylene test tubes
  • Polytron homogenizer
  • Glass fiber filters (GF/C type, Whatman)
  • Cell harvester (Brandel Instruments)
  • -scintillation counter
  • Curve fitting programs (e.g., KELL, Biosoft; or Prism, GraphPad Software)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 1.25.1
    (A) Saturation binding of [3H]PN200-100 to L-type calcium channels in mouse heart membrane preparation (n = 2). (B) Scatchard analysis of the specific binding data: Kd = 54.9 pM and Bmax = 116.4 fmol/mg protein.

    View Image
  • Figure 1.25.2
    Inhibition of [3H]PN200-100 binding in mouse heart membrane preparation by nitrendipine and nifedipine (n = 2). The IC50 values are 408 and 728 pM for nitrendipine and nifedipine, respectively (Ki(nitrendipine) = 206 pM and Ki(nifedipine) = 367 pM).

    View Image
  • Figure 1.25.3
    (A) Saturation binding of [125I] -conotoxin MVIIA to N-type calcium channels in rat brain membrane preparation (n = 2). (B) Scatchard analysis of the specific binding data: Kd = 17.9 pM and Bmax = 1503 fmol/mg protein.

    View Image
  • Figure 1.25.4
    Inhibition of [125I] -conotoxin MVIIA binding to rat brain membrane preparation by -conotoxin MVIIA (n = 2). The IC50 value is 41.2 pM (Ki = 18.9 pM).

    View Image

Literature Cited

Literature Cited
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    Cheng, Y.C. and Prusoff, W.H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition of an enzyme reaction. Biochem.Pharmacol 23:3099-3108.
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    Lee, J.H., Daud, A.N. Cribbs, L.L., Lacerda, A.E., Pereverzev, A., Klockner, U., Schneider, T., and Perez-Reyes, E. 1999. Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J. Neurosci. 19:1912-21
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    Mori, Y., Friedrich, T., Kim, M.-H., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi., T., Mikoshiba, K., Imoto, K., Tanabe, T., and Numa, S. 1991. Primary structure and functional expression from complementary DNA of a calcium channel. Nature 350:398-402.
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