Translational activation and repression by distinct elements within the 5'-UTR of ENaC alpha-subunit mRNA

The rat amiloride-sensitive epithelial Na(+) channel (rENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits, alpha, beta, and gamma. We hypothesized that alpha-rENaC translation is regulated via its 5'-untranslated region (UTR). Transient transfections of alpha-rENaC promoter-reporter constructs in representative epithelial cell lines demonstrated up to fivefold differences in activity among constructs containing different amounts of the alpha-rENaC 5'-UTR sequence. Differences in reporter protein activity did not parallel differences in reporter mRNA, demonstrating that 5'-UTR regulation must be at the level of translation. Specifically, translation was enhanced by a region extending from +53 to +211 bp downstream from the transcription start site and repressed by the region between +367 and +499 bp. Examination of the 5'-UTR sequence revealed an out-of-frame initiation codon within the repressive region, 43 bp upstream from the start of the alpha-rENaC open reading frame. Mutational analysis of this upstream start codon indicated that it plays, at most, a minor role in impeding translation both in vitro and in vivo, suggesting that additional mechanisms of translational regulation are operative.