A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α728. The baseline whole cell conductance of oocytes expressing trimeric α728βγ channels was 898.1±277.2 and 49.59±13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α669βγ in same solutions. α728βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α669βγ in low and high Na+ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na+]i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α728βγ was likely mediated by enhanced channel activity or open probability. Expression of α728 in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.

The Alpha subunit of ENaC is critical to the ability to form a channel. Alpha‐like subunits have been described and have been shown to affect channel activity and/or regulation. A new isoform of the alpha subunit containing an extended N‐terminus resulting in a 728 a.a. subunit was recently identified. We examined the electrophysiological properties of channels formed by this isoform. Channels formed by α728βγ exhibited much higher activity than those formed by α669βγ in the Xenopus oocyte expression system. These differences were much larger under conditions which elevated the intracellular [Na+]. α728βγ also exhibited reduced current/voltage rectification. These differences occurred despite similar single channel conductances (~5.5 pS). The role of intracellular Na+ and Na+ self‐inhibition in these differences was examined. We find that α728βγ sustained much higher activity under elevated [Na+] than α669βγ and that intracellular and acute increase of [Na+] by injection of Na2SO4 led to further increase of conductance. α728βγ channels were further activated by extracellular cleavage by trypsin or subtilisin; with 2 notable differences: 1) fold activation was smaller than that observed with α669βγ, and 2) the large baseline activity of α728βγ channels was observed in the absence of detectable cleavage at the plasma membrane. These data indicate that α728 forms the structural basis of an ENaC with differing activity and regulation than α669. Grant Funding Source: National Institute of Diabetes and Digestive and Kidney Diseases Grant DK‐55626