Purification of a K+-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

Toru Ide, Takuma Morita, Takashi Kawasaki, Takahisa Taguchi, Michiki Kasai

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A K+ channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K+ channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca2+-ATPase antibody did not recognize the protein. The characteristics of the K+ channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.

Original languageEnglish
Pages (from-to)213-220
Number of pages8
JournalBBA - Biomembranes
Volume1067
Issue number2
DOIs
Publication statusPublished - Aug 26 1991
Externally publishedYes

Fingerprint

Lipid bilayers
Lipid Bilayers
Sarcoplasmic Reticulum
Purification
Chromatography
Framycetin
Gels
Affinity chromatography
Proteins
Dialysis
Calcium-Transporting ATPases
Concanavalin A
Affinity Chromatography
Silver
Gel Chromatography
Anions
Polyacrylamide Gel Electrophoresis
Ion exchange
Antibodies
Electric potential

Keywords

  • Lipid bilayer method
  • Potassium ion channel
  • Reconstituted lipid vesicle
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Purification of a K+-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system. / Ide, Toru; Morita, Takuma; Kawasaki, Takashi; Taguchi, Takahisa; Kasai, Michiki.

In: BBA - Biomembranes, Vol. 1067, No. 2, 26.08.1991, p. 213-220.

Research output: Contribution to journalArticle

Ide, Toru ; Morita, Takuma ; Kawasaki, Takashi ; Taguchi, Takahisa ; Kasai, Michiki. / Purification of a K+-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system. In: BBA - Biomembranes. 1991 ; Vol. 1067, No. 2. pp. 213-220.
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