Syntaxin 1A Regulates ENaC via Domain-specific Interactions

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 α-, β-, and γ-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 α-, β-, or γ-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 β-subunit PY motif increases I Na by interfering with ENaC endocytosis. In contrast to subunit truncation, a β-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.