Question: 

As part of the SNARE complex, syntaxins are involved in vesicle fusion at the plasma membrane. Thus, syntaxins may play a role in ion channel trafficking. Previously, it has been reported that syntaxin 1A inhibits the epithelial sodium channel (ENaC) heterologously expressed in oocytes^1,2 by reducing channel expression at the cell surface². In contrast, syntaxin 3 (STX3) has been shown to stimulate ENaC¹. The aim of the present study was to explore a possible effect of syntaxin 4 (STX4) on ENaC function and to investigate the mechanism underlying the stimulatory effect of STX3.

Methodology: 

Rat ENaC alone or together with rat STX3 or STX4 were expressed in Xenopus laevis oocytes and amiloride(2mM)-sensitive whole-cell currents (DI_ami) were detected using the two-electrode voltage-clamp technique. In addition, ENaC was proteolytically activated with chymotrypsin (2 mg/ml) in the presence and absence of STX3 or STX4. N indicates the number of batches of oocytes, n indicates the number of individual oocytes used.

Results: 

Coexpression of ENaC and STX3 (n=129, N=13) increased DI_ami by ~100% compared to oocytes expressing ENaC alone (n=130, p'0.001), which is in agreement with previous results¹. Application of chymotrypsin on oocytes expressing ENaC and STX3 stimulated DI_ami by ~4.7fold (n=27, N=4). This effect was not significantly different from matched oocytes expressing ENaC alone (~4.1fold, n=27, p=0.283). Importantly, following activation with chymotrypsin, DI_ami of oocytes expressing ENaC and STX3 was ~100% higher compared to that of oocytes expressing ENaC alone. Coexpression of ENaC and STX4 (n=65, N=6) increased DI_ami by ~90% compared to matched oocytes expressing ENaC alone (n=65, p'0.001). Chymotrypsin stimulated DI_ami of oocytes expressing ENaC and STX4 by ~4.2fold (n=29, N=3). This effect was not significantly different from the effect on matched oocytes expressing ENaC alone (~3.6fold, n=29, p=0.147). Importantly, following activation with chymotrypsin, DI_ami of oocytes expressing ENaC and STX4 was ~80% higher compared to that of oocytes expressing ENaC alone.

Conclusion: 

We confirmed the stimulatory effect of STX3 on ENaC and demonstrated for the first time that STX4 also stimulates ENaC in oocytes. The degree of proteolytic ENaC activation by chymotrypsin was similar in oocytes expressing ENaC alone or coexpressing ENaC and STX3 or STX4. This suggests that both syntaxins stimulate ENaC predominantly by increasing channel expression at the cell surface.

References: 1. Saxena et al. 1999, JBC 274:20812-7; 2. Qi et al. 1999, JBC 274:30345-8