Syntaxin 1A regulates ENaC channel activity.
Na(+) entry across the apical membranes of many absorptive epithelia is determined by the number (N) and open probability (P(o)) of epithelial sodium channels (ENaC). Previous results showed that the H3 domain of syntaxin-1A (S1A) binds to ENaC to reduce N, supporting a role for S1A in the regulation of ENaC trafficking. The aim of this study was to determine whether S1A-induced reductions in ENaC current also result from interactions between cell surface ENaC and S1A that alter ENaC P(o). Injection of a glutathione S-transferase (GST)-H3 S1A fusion protein into ENaC-expressing Xenopus oocytes inhibited whole cell Na(+) current (I(Na)) by 33% within 5 min. This effect was dose-dependent, with a K(i) of 7 ng/microl (approximately 200 nm). In contrast, injection of GST alone or a H3 domain-deleted GST-S1A fusion protein had no effect on I(Na). In cell-attached patch clamp experiments, GST-H3 acutely decreased ENaC P(o) by 30%, whereas GST-S1A Delta H3 was without effect. Further analysis revealed that ENaC mean closed time was significantly prolonged by S1A. Interestingly, GST-H3 had no effect on channel activity of an ENaC pore mutant that constitutively gates open (P(o) approximately equal 1.0), supporting the idea that S1A alters the closed state of ENaC and indicating that the actions of S1A on ENaC trafficking and gating can be separated experimentally. This study indicates that, in addition to a primary effect on ENaC trafficking, S1A interacts with cell surface ENaC to rapidly decrease channel gating. This rapid effect of S1A may modulate Na(+) entry rate during rapid increases in ENaC N.
Condliffe
Steven B
SB
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15217, USA.
Zhang
Hui
H
Frizzell
Raymond A
RA
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D021381:Protein Transport; D018075:RNA, Complementary / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D015222:Sodium Channels / Q000096:biosynthesis* / Q000378:metabolism; D050827:Syntaxin 1; D013997:Time Factors; D014981:Xenopus
D000954:Antigens, Surface; D053503:Epithelial Sodium Channels; D009419:Nerve Tissue Proteins; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; C494214:Stx1a protein, mouse; D050827:Syntaxin 1; D012964:Sodium; D005982:Glutathione Transferase