Revisiting the Heteromultimeric Structure of ENaC in XENOPUS Leavis Oocytes

Abstract

The functional epithelial sodium channel (ENaC) is a heteromeric channel formed by three homologous alpha, beta and gamma subunits.Several functional and biochemical studies have provided evidence that the ENaC is a heterotetramer formed by 2alpha, 1beta and 1gamma subunits. Recently, a high-resolution crystal structure has been obtained from an ortholog of ENaC, the acid-sensing ion channel ASIC1, which showed a homotrimeric channel. This discrepancy between two channels of the same ion channel family, motivated us to revisit the subunit oligomerization of ENaC.His-tagged ENaC alpha, beta and gamma subunits were expressed in Xenopus leavis oocytes. The three ENaC subunits can be co-purified on Ni+2-NTA agarose beads in an alpha beta gamma-ENaC complex. On Western blot, the ENaC subunits showed typical post-translation modifications associated with a functional channel. Using differentially tagged ENaC subunits, we investigated whether the ENaC complex contains more than a single alpha, beta or gamma subunits. Two differentially tagged alpha subunits co-purified with beta and gamma subunits, indicating that ENaC is formed by more than one alpha subunit. The purified ENaC complex channel eluted on Sephadex G 200 column in a fraction corresponding to a molecular weight of 350 kDa, which was higher than expected for a alpha beta gamma-ENaC.These data confirm previous reports that the functional ENaC channel is a heterotetramer made of 2alpha 1beta, and 1gamma subunits.