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Int J Biol Sci
2006 Nov 06;31:47-56. doi: 10.7150/ijbs.3.47.
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Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells.
Saxena SK
,
Singh M
,
Kaur S
,
George C
.
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The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distalnephron 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 gammaENaC 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.
Fig 1. Syntaxin1A augments currents - HT-29 cells grown on cell inserts were transfected with wild-type syntaxin1A (Syn1A) and its truncated constructs. Inset- Structure of syntaxin1A. TMD represents the transmembrane domain. 48 hours later the amiloride-sensitive currents were recorded as described in the text. The data represents a mean of five individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.
Fig 2. Syntaxin1A up-regulates amiloride-sensitive currents in a dose-dependent manner - HT-29 cells grown on cell inserts were transfected with wild-type syntaxin1A (Syn1A) at different concentrations. 48 hours later amiloride-sensitive currents were recorded (A) as described in the text. (B) The protein extracts from the transfected cells were solubilized and analyzed by Western blot analysis. The blots were developed with syntaxin1A antibody. The data represents a mean of three individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.
Fig 3. Syntaxin1A antibody inhibits amiloride-sensitive currents - The HT-29 cells were targeted with anti-syntaxin, or anti-ENaC antibodies or irrelevant IgG using the Chariot® protein delivery system. Antibodies were complexed with the Chariot® reagent at a ratio of 1 ng IgG: 2 μL Chariot® in 100 μL PBS for 30 min. Then the IgG: Chariot® complexes were overlaid onto cultured cells in the presence of fresh serum free culture medium for 3 hr and amiloride-sensitive currents were measured. Data represent a mean of three individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks.
Fig 4. ENaC interacts with different syntaxin1A domains - Endogenous ENaC interacts with syntaxin in HT-29 cells - GST-syntaxin proteins were used to pull down proteins bound to natively expressed ENaC in HT-29 cell lysates. The immune complex was adsorbed on glutathione beads and then separated by SDS-Polyacrylamide gel electrophoresis and transferred to PVDF membrane. The blots were probed with affinity purified subunit specific γENaC antibody. The data shows interaction of multiple syntaxin1A domains with ENaC and points to its involvement and physiological significance in the regulation of the amiloride-sensitive epithelial sodium channel in native cells.
Fig 5. Syntaxin1A domains modulate ENaC expression at the cell surface - HT-29 cells were transfected with wild-type syntaxin1A and its truncated constructs. Cell surface proteins were biotinylated with Sulpho-NHS-SS-biotin, pulled down with streptavidin-agarose separated by SDS-PAGE and transferred to PVDF membrane. The blots were probed with γENaC antibody (A). The protein was analyzed by densitometry (B). The data reflect increased expression of ENaC in HT-29 cells transfected with syntaxin1A, which is reflected in enhanced amiloride-sensitive currents reported in the text. Data represent three individual experiments each performed with different batches of HT-29 cells.
Fig 7. Syntaxin1A stimulates amiloride-sensitive channel (ENaC) activity by multiple signaling modules - HT-29 cells (60-70% confluent) on cell inserts were transfected with wild-type syntaxin1A (Syn1A) and incubated in the presence of indicated inhibitors; PD98059 (PD; 50 μM) or LY294002 (LY; 50 μM), RpCPTcAMP (RpcAMP; 30 μM) and PMA (200 nM) 48 hours later. The inhibition or activation in intact cells was measured with the indicated concentration of reagents or dimethyl sulfoxide carrier (0.1%, v/v) for 45 min at 37°C. The amiloride-sensitive currents were recorded as described in the text. The data represents a mean of three individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.
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