Abstract
The epithelial sodium channel (ENaC) is a heterotrimeric protein responsible for Na(+) absorption across the apical membranes of several absorptive epithelia. The rate of Na(+) absorption is governed in part by regulated membrane trafficking mechanisms that control the apical membrane ENaC density. Previous reports have implicated a role for the t-SNARE protein, syntaxin 1A (S1A), in the regulation of ENaC current (I(Na)). In the present study, we examine the structure-function relations influencing S1A-ENaC interactions. In vitro pull-down assays demonstrated that S1A directly interacts with the C termini of the alpha-, beta-, and gamma-ENaC subunits but not with the N terminus of any ENaC subunit. The H3 domain of S1A is the critical motif mediating S1A-ENaC binding. Functional studies in ENaC expressing Xenopus oocytes revealed that deletion of the H3 domain of co-expressed S1A eliminated its inhibition of I(Na), and acute injection of a GST-H3 fusion protein into ENaC expressing oocytes inhibited I(Na) to the same extent as S1A co-expression. In cell surface ENaC labeling experiments, reductions in plasma membrane ENaC accounted for the H3 domain inhibition of I(Na). Individually substituting C terminus-truncated alpha-, beta-, or gamma-ENaC subunits for their wild-type counterparts reversed the S1A-induced inhibition of I(Na), and oocytes expressing ENaC comprised of three C terminus-truncated subunits showed no S1A inhibition of I(Na). C terminus truncation or disruption of the C terminus beta-subunit PY motif increases I(Na) by interfering with ENaC endocytosis. In contrast to subunit truncation, a beta-ENaC PY mutation did not relieve S1A inhibition of I(Na), suggesting that S1A does not perturb Nedd4 interactions that lead to ENaC endocytosis/degradation. This study provides support for the concept that S1A inhibits ENaC-mediated Na(+) transport by decreasing cell surface channel number via direct protein-protein interactions at the ENaC C termini.
Highlights
The epithelial Naϩ channel (ENaC)1 is located at the apical membranes of aldosterone-responsive epithelial cells of the
We reasoned that the ENaC cytoplasmic domains would be the potential binding sites for syntaxin 1A (S1A), a type 2 plasma membrane protein that has no significant structure within the extracellular compartment
As in similar protein interaction studies, a truncated syntaxin 1A (S1A4–267) lacking the transmembrane domain was employed for its enhanced solubility and purification properties and because the last 21 amino acids of the S1A C terminus constitute the transmembrane domain, which is not expected to be involved in cytosolic protein-protein interactions
Summary
The epithelial Naϩ channel (ENaC) is located at the apical membranes of aldosterone-responsive epithelial cells of the. Saxena et al [15] found that S1A co-precipitated from Xenopus oocytes with FLAG-tagged subunits of both - and ␥-ENaC, whereas possible interactions with ␣-ENaC were not addressed These studies have identified a functional interaction of ENaC with S1A, the precise domains of both proteins involved in these interactions remain unclear. Our results define the domain interactions between S1A and the ENaC subunits and demonstrate reversal of the S1A inhibitory action on INa by elimination of these domains These findings, coupled with the effects of S1A on ENaC cell surface expression and on ENaC bearing a PY mutation, support the concept that the S1A-ENaC interaction controls ENaC trafficking mediated by SNARE protein interactions
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