Question: 

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is known to inhibit the epithelial sodium channel (ENaC) which consists of three subunits (a,b,g). In humans, an additional dENaC-subunit exists which is thought to assemble into dbgENaC channels. The aim of this study was to investigate the effect of CFTR on dbgENaC function.

Methodology: 

Human dbgENaC and CFTR were expressed in Xenopus laevis oocytes. dbgENaC function was assessed by measuring the amiloride(100mM)-sensitive whole-cell current (DI_ami) with the two-electrode voltage-clamp technique. Average open probability (Po) of ENaC was determined using the mutant bS520C-ENaC which can be converted by exposure to MTSET (1mM) to a channel with a Po close to one. CFTR function was assessed as CFTR(Inh)-172(5mM)-sensitive whole-cell current (DI_CFTR172) after activation of CFTR with IBMX (1mM) and forskolin (1mM).

Results: 

Co-expression of dbgENaC and CFTR stimulated DI_CFTR172 by ~70% compared to oocytes expressing CFTR alone. In contrast, without activation of CFTR, co-expression of dbgENaC and CFTR reduced DI_ami by ~50% compared to oocytes expressing dbgENaC alone. Interestingly, application of IBMX/forskolin increased DI_ami by ~50% in both oocytes expressing dbgENaC alone or co-expressing dbgENaC with CFTR. Proteolytic activation of dbgENaC by chymotrypsin (2mg/ml) stimulated DI_ami 2.4fold in dbgENaC expressing oocytes and 2.9fold in oocytes co-expressing dbgENaC and CFTR. This indicates a lower average Po for dbgENaC in the presence of CFTR. Using the mutant bS520C-ENaC as a tool, we estimated an average Po for dbgENaC of ~0.5 in the absence and of ~0.4 in the presence of CFTR.

Conclusion: 

Similar to abgENaC, dbgENaC enhances CFTR function but is inhibited by inactive CFTR. In contrast to abgENaC, dbgENaC is not inhibited by CFTR activation. The inhibitory effect of co-expressed CFTR on baseline DI_ami is in part caused by a reduced average Po. In agreement with this result, the relative stimulatory effect of chymotrypsin on dbgENaC is slightly larger in the presence of CFTR than in its absence. This suggests that CFTR may reduce the constitutive proteolytic activation of dbgENaC by endogenous proteases. Interestingly, in contrast to abgENaC, dbgENaC can be stimulated by a brief (~15 min) exposure of the oocytes to IBMX/forskolin. In conclusion our results demonstrate that CFTR alters dbgENaC function in a slightly different manner than the function of abgENaC. In humans this may be relevant for tissue specific regulation of ENaC activity. The molecular mechanisms involved in the observed functional interaction of dbgENaC and CFTR remain to be determined.