Number of subunits comprising the epithelial sodium channel

The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, alpha, beta, and gamma. Understanding the struc ture and function of this channel and its abnormal behavior in disease requ ires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological metho ds to evaluate the number of subunits in the ENaC complex expressed in Xeno pus laevis oocytes, In oocytes expressing wild-type hENaC (alpha, beta, and gamma subunits), clusters of particles appeared in the protoplasmic face o f the plasma membranes. The total number of particles in the clusters was c onsistent with the whole-cell amiloride-sensitive current measured in the s ame cells. The size frequency histogram for the particles in the clusters s uggested the presence of an integral membrane protein complex composed of 1 7 +/- 2 transmembrane alpha-helices. Because each ENaC subunit has two puta tive transmembrane helices, these data suggest that in the oocyte plasma me mbrane, the ENaC complex is composed of eight or nine subunits. At high mag nification, individual ENaC particles exhibited a near-square geometry. Fun ctional studies using wild-type alpha beta-hENaC coexpressed with gamma-hEN aC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and. cadmium, suggested that the functiona l channel complex contains more than one gamma subunit, These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are gamma subunits.