Abstract
Epithelial Na(+) channel (ENaC) function is regulated by the intracellular Na(+) concentration ([Na(+)]i) through a process known as Na(+) feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na(+)]i alters ENaC cleavage. We show here that [Na(+)]i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na(+)]i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na(+)]i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na(+)]i. The hypothesis that [Na(+)]i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na(+)]i. Therefore, increased [Na(+)]i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.
Highlights
Epithelial Na؉ channel (ENaC) activity decreases when intracellular Naϩ is elevated
Epithelial Na؉ channel (ENaC) function is regulated by the intracellular Na؉ concentration ([Na؉]i) through a process known as Na؉ feedback inhibition
Fisher rat thyroid (FRT) cells were transfected with ␣␥ ENaC and cultured in media with and without 50 M amiloride to manipulate [Naϩ]i
Summary
ENaC activity decreases when intracellular Naϩ is elevated. Results: High intracellular Naϩ inhibits N-glycan remodeling and prevents proteolytic processing by endogenous and exogenous proteases. Likewise, [Naϩ]i seems to regulate the open probability of the channel [9] In this regard, recent evidence suggests that intracellular Naϩ regulates the cleavage state of ENaC so that the channel undergoes increased proteolytic activation when [Naϩ]i is low [10, 11]. Hughey et al [12,13,14] has demonstrated that ENaC maturation involves modification of immature N-glycans to complex mature oligosaccharides and proteolytic processing of the ␣ and ␥ ENaC subunits by furin [12,13,14] (Fig. 1) Because these two modifications occur together in an “all or none event,” we hypothesized that [Naϩ]i reduces the proportion of channels that undergo maturation in the Golgi and trans-Golgi network.
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