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 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.
Condliffe
Steven B
SB
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Carattino
Marcelo D
MD
Frizzell
Raymond A
RA
Zhang
Hui
H
D000818:Animals; D000954:Antigens, Surface / Q000502:physiology*; D001665:Binding Sites; D003001:Cloning, Molecular; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D009419:Nerve Tissue Proteins / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000378:metabolism; D018075:RNA, Complementary / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics* / Q000502:physiology; D050827:Syntaxin 1; D014158:Transcription, Genetic
D000954:Antigens, Surface; D053503:Epithelial Sodium Channels; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C494212:STX1A protein, human; D015222:Sodium Channels; C494214:Stx1a protein, mouse; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D012964:Sodium