Characterization of an amiloride binding region in the α-subunit of ENaC

Abstract

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the α-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278–283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type α-subunits or α-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities ( Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high- Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type α-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.