Characterization of Calcium Channel Binding

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Abstract
Table of Contents
Materials
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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 [³ H]PN200?100 and [¹²⁵ I](conotoxin MVIIA. Voltage?dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain

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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
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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
[³ H]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
[¹²⁵ I]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

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

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Figure 1.25.2 Inhibition of [³ H]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 ( K _{i(nitrendipine)} = 206 pM and K _{i(nifedipine)} = 367 pM).

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Figure 1.25.3 (A ) Saturation binding of [¹²⁵ I] ω‐conotoxin MVIIA to N‐type calcium channels in rat brain membrane preparation ( n = 2). (B ) Scatchard analysis of the specific binding data: K _d = 17.9 pM and B _max = 1503 fmol/mg protein.

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Figure 1.25.4 Inhibition of [¹²⁵ I] ω‐conotoxin MVIIA binding to rat brain membrane preparation by ω‐conotoxin MVIIA ( n = 2). The IC₅₀ value is 41.2 pM ( K _i = 18.9 pM).

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Literature Cited

Literature Cited
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Characterization of Calcium Channel Binding

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited