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-in activated Botulinum toxin A, reversed the inhibitory effect of SNAP-23 on amiloride-sensitive currents. However, syntaxin 1A and SNA-P-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 alpha ENaC. 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. (c) 2006 Elsevier Inc. All rights reserved.