The Epithelial Na⁺ channel (ENaC) is the rate-limiting step for sodium reabsorption at the distal renal tubule. This channel is expressed at the plasma membrane as a multimeric complex of three subunits alpha, beta and gamma. Nevertheless, the number of subunits composing functional channels is controversial. It has been proposed that ENaC is formed with a tetrameric arrangement of 2alpha:1beta:1gamma (Firsov et al 1998, EMBO J 17: 344; Kosari et al 1998, JBC 273: 13469) but other authors suggest a higher-ordered stoichiometry of 3alpha:3beta:3gamma (Staruschenko et al 2004, JBC 279:27729).

Using fluorescently tagged ENaC subunits and fluorescence resonance energy transfer (FRET) we are exploring the subunit composition of these channels at the cell membrane. To that end, we have generated the fusion proteins of alpha, beta and gamma ENaC subunits with fluorescent proteins yellow (YFP) or cyan (CFP). We are expressing various combinations of this fluorescent subunits in Xenopus oocytes and studying the fluorescence emission spectra at the membrane using a spectral confocal microscope or Total Internal Reflection Fluorescence Microscopy (TIRFM) coupled to a spectrograph.