Abstract
The epithelial Na(+) channel (ENaC) has three subunits; the expression of each can be regulated. Liddle's syndrome is caused by an activating mutation in the C terminus of either the beta or gamma subunit. We used a doxycycline-regulated adenovirus system to express varying levels of human gammaENaC in renal collecting duct (M1 cell) monolayers. Increasing levels of wild type human gamma ENaC (gammahENaC) produced a 2.5-fold enhancement of Na(+) transport. Expression of a truncated C terminus produced less protein than wild type or a gammaY627A missense mutation. However, either of these mutations produced a approximately 4-fold increase in Na(+) transport despite the different levels of protein expression. Unexpectedly, overexpression of a marginally detectable amount of gammahENaC was sufficient to produce a full increase in Na(+) transport; a further increase in protein expression produced no further increase in Na(+) transport. Steroid treatment increased Na(+) transport to a similar absolute magnitude in control monolayers and in monolayers expressing all types of gammahENaC. Withdrawal of steroids after 24 h produced a decline in Na(+) transport over 8 h in monolayers expressing wild type but not the Liddle's mutation. Using treatment with brefeldin A to estimate the disappearance rate constants, we found progressively slower disappearance rates in monolayers overexpressing gammahENaC and the Liddle's mutant. Calculated insertion rates were slower for the Liddle's mutant than for wild type despite increasing rates of Na(+) transport. These results raise questions regarding previously held assumptions about the behavior of ENaC.