The epithelial Na+ channel (ENaC) possesses a large extracellular domain formed by a ?-strand core enclosed by three peripheral ?-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (?, ?, or ?). Removing the ? subunit thumb domain had no effect on Na+ currents when expressed in Xenopus oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the ? or ? subunit thumb domain exhibited significantly reduced Na+ currents along with a large reduction in channel surface expression. Moreover, channels lacking an ? or ? thumb domain exhibited a diminished Na+ self-inhibition response, whereas this response was retained in channels lacking a ? thumb domain. In turn, deletion of the ? thumb domain had no effect on the degradation rate of the immature ? subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature ? subunit and mature ? subunit was observed when the ? or ? thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the ? and ? thumb domains both have important roles in the channel`s inhibitory response to external Na+. Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity.