Characterization of Calcium Channel Binding

Wei Zheng1

1 Merck 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

  • 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]ω‐Conotoxin‐MVIIA Saturation Binding to N‐type Calcium Channels in Rat Brain Membranes
  • Basic Protocol 4: Measurement of Compound and [125I]ω‐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
  • β‐scintillation counter
  • Curve fitting programs (e.g., KELL, Biosoft; or Prism, GraphPad Software)

Basic Protocol 2: Measurement of [3H]PN200‐100 Displacement Binding to L‐type Calcium Channels in Cardiac 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]ω‐conotoxin‐MVIIA (NEN Life Sciences)
  • ω‐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)
  • γ‐scintillation counter
  • Curve fitting programs (e.g., KELL, Biosoft; or Prism, GraphPad Software)
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Figures

  •   FigureFigure 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.
  •   FigureFigure 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).
  •   FigureFigure 1.25.3 (A) Saturation binding of [125I] ω‐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.
  •   FigureFigure 1.25.4 Inhibition of [125I] ω‐conotoxin MVIIA binding to rat brain membrane preparation by ω‐conotoxin MVIIA ( n = 2). The IC50 value is 41.2 pM ( Ki = 18.9 pM).

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Literature Cited

Literature Cited
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