Epithelial sodium channel is regulated by SNAP-23/syntaxin 1A interplay.
Sodium-selective amiloride-sensitive epithelial channel (ENaC) located in the apical membrane is involved in the reabsorption of sodium in tight epithelia. The soluble N-ethylmaleimide-sensitive attachment receptors (SNAREs) mediate vesicle trafficking in a variety of cell systems. Syntaxin (a t-SNARE) has been shown to interact with and functionally regulate a number of ion channels including ENaC. In this study, we investigated the role of SNAP-23, another SNARE protein, on ENaC activity in the HT-29 colonic epithelial cell system and Xenopus oocytes. Recording of amiloride-sensitive currents in both systems suggest that SNAP-23 modulates channel function, though a much higher concentration is required to inhibit ENaC in Xenopus oocytes. The introduction of Botulinum toxin A (a neurotoxin which cleaves SNAP-23), but not Botulinum toxin B or heat-inactivated Botulinum toxin A, reversed the inhibitory effect of SNAP-23 on amiloride-sensitive currents. However, syntaxin 1A and SNAP-23 combined portray a complex scenario that suggests that this channel interacts within a quaternary complex. Synaptotagmin expression neither interacts with, nor showed any effect on amiloride-sensitive currents when co-expressed with ENaC. Pull down assays suggest mild interaction between ENaC and SNAP-23, which gets stronger in the presence of syntaxin 1A. Data further suggest that SNAP-23 possibly interacts with the N-terminal alphaENaC. These functional and biochemical approaches provide evidence for a complex relationship between ENaC and the exocytotic machinery. Our data suggest that SNARE protein interplay defines the fine regulation of sodium channel function.
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA. ssaxena@stevens.edu
Sunil K
SK
Saxena
Constantine M
CM
George
Vadim
V
Pinskiy
Blake
B
McConnell
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002460:Cell Line; D004847:Epithelial Cells; D053503:Epithelial Sodium Channels; D006801:Humans; D015640:Ion Channel Gating; D009865:Oocytes / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050766:Qb-SNARE Proteins / Q000502:physiology*; D050767:Qc-SNARE Proteins / Q000502:physiology*; D051381:Rats; D015222:Sodium Channels / Q000502:physiology*; D050857:Synaptotagmins / Q000378:metabolism; D050827:Syntaxin 1 / Q000502:physiology*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D015222:Sodium Channels; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D050857:Synaptotagmins; D000584:Amiloride; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A