Biophysical approach to determine the subunit stoichiometry of the epithelial sodium channel using the Xenopus laevis oocyte expression system.
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.
Kosari
Farhad
F
Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA.
Sheng
Shaohu
S
Kleyman
Thomas R
TR
D000818:Animals; D004566:Electrodes; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018075:RNA, Complementary / Q000235:genetics; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D018075:RNA, Complementary