Reconstitution of expressed K(Ca) channels from Xenopus oocytes to lipid bilayers

G. Perez, A. Lagrutta, John Adelman, L. Toro

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Abstract

Reconstitution of large conductance calcium-activated potassium (K(Ca)) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, K(Ca) channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo K(Ca) channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native K(Ca) channels. They possess a mean unitary conductance of ≃260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 μM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo K(Ca) channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (K(D) = 158 μM, at 0 mV) and internal Ba2+ (K(D) = 76 μM, at 40 mV). In addition, they were blocked by internally applied 'ball' inactivating peptide (K(D) = 480 μM, at 40 mV). These results demonstrate that cloned K(Ca) channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.

Original languageEnglish (US)
Pages (from-to)1022-1027
Number of pages6
JournalBiophysical Journal
Volume66
Issue number4
StatePublished - 1994
Externally publishedYes

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Lipid Bilayers
Xenopus
Oocytes
Calcium
Potassium
Charybdotoxin
Tetraethylammonium
Membrane Lipids
Drosophila
Cell Membrane
Pharmacology
Ions
Peptides
Genes

ASJC Scopus subject areas

  • Biophysics

Cite this

Perez, G., Lagrutta, A., Adelman, J., & Toro, L. (1994). Reconstitution of expressed K(Ca) channels from Xenopus oocytes to lipid bilayers. Biophysical Journal, 66(4), 1022-1027.

Reconstitution of expressed K(Ca) channels from Xenopus oocytes to lipid bilayers. / Perez, G.; Lagrutta, A.; Adelman, John; Toro, L.

In: Biophysical Journal, Vol. 66, No. 4, 1994, p. 1022-1027.

Research output: Contribution to journalArticle

Perez, G, Lagrutta, A, Adelman, J & Toro, L 1994, 'Reconstitution of expressed K(Ca) channels from Xenopus oocytes to lipid bilayers', Biophysical Journal, vol. 66, no. 4, pp. 1022-1027.
Perez, G. ; Lagrutta, A. ; Adelman, John ; Toro, L. / Reconstitution of expressed K(Ca) channels from Xenopus oocytes to lipid bilayers. In: Biophysical Journal. 1994 ; Vol. 66, No. 4. pp. 1022-1027.
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