Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both γENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.

Reconstitution and Characterization of a Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-sensitive Ca2+ Release Channel from Liver Lysosomes of Rats

“…This suggests that TRP-ML1 may be the identity of lysosomal NAADP-sensitive Ca 2ϩ release channels. In regard to TRP-ML1 channel blockers, several studies have used amiloride for characterization of TRP-ML1 channel activity, although it has often been used as a sodium channel blocker, such as epithelial Na ϩ channel (ENaC) (18,52,53). Interestingly, this compound substantially blocked NAADP-induced activation of the channels reconstituted in the lipid bilayer.…”

Regulation of αENaC Transcription

“…Furin-mediated cleavage of α- and γENaC activates the channels by relieving Na + self-inhibition (Sheng et al, 2006). The vesicle traffic regulatory protein syntaxin 1A contains distinct inhibitory and stimulatory domains that interact with ENaC subunits and determine the overall ENaC functionality/regulation under distinct physiological conditions (Condliffe, Zhang, & Frizzell, 2004; Qi et al, 1999; Saxena, Singh, Kaur, & George, 2007). However, the aldosterone effect on furin or syntaxin 1A expression has not been documented yet.…”

Organization of the ENaC-regulatory machinery

“…Instead, S-1A was found to interact with ENaC C-terminal tails, and shown to influence surface ENaC levels via direct protein–protein interactions at the C-termini (Condliffe et al , 2003). Studies done in HT-29 cells, a physiologically relevant cell line that expresses both endogenous ENaC and S-1A, and also responds to aldosterone stimulation, suggested that ENaC interacts with multiple domains of S-1A, which differentially regulate channel function (Saxena et al , 2007). It appears that S-1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits and regulate its overall activity under physiological conditions, in association with another SNARE protein, SNAP-23 (Saxena et al , 2006).…”

“…SNAP-23 is believed to interact with the N-terminus of α-ENaC, within a quaternary complex that includes S-1A, and modulate channel activity. S-1A-mediated regulation of ENaC involves multiple mechanisms including the PKA-, PLC-, PI3K-, and ERK1/2-signaling pathways, again, suggesting the assembly of ENaC-regulatory signals at or near the plasma membrane (Saxena et al , 2007). …”