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.
Saxena
Sunil K
SK
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA. ssaxena@stevens.edu
George
Constantine M
CM
Pinskiy
Vadim
V
McConnell
Blake
B
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