Biophysical approach to determine the subunit stoichiometry of the epithelial sodium channel using the Xenopus laevis oocyte expression system.

Farhad Kosari, Shaohu Sheng, Thomas R. Kleyman

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Abstract

The amiloride-sensitive epithelial Na+ channel (ENaC) is typically composed of three structurally related subunits termed alpha, beta, and gamma. We describe methods to determine the functional subunit stoichiometry of ENaC based on a biophysical approach that was first introduced in 1991 to determine the subunit stoichiometry of a voltage-gated K+ channel. The strategy is to analyze channel sensitivity to a specific blocker when various mixtures of block-sensitive and blocker-insensitive subunits are coexpressed in a heterologous expression system. Details related to the expression of wild type and mutant ENaCs in Xenopus oocytes and the examination of blocker sensitivity by two-electrode voltage clamp, as well as analysis of data are provided.

Original languageEnglish (US)
Pages (from-to)53-63
Number of pages11
JournalMethods in molecular biology (Clifton, N.J.)
Volume337
StatePublished - 2006

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Epithelial Sodium Channels
Xenopus laevis
Oocytes
Voltage-Gated Potassium Channels
Amiloride
Xenopus
Electrodes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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