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.
Highlights
The epithelial Na؉ channel (ENaC) has three subunits; the expression of each can be regulated
Overexpression of a marginally detectable amount of ␥hENaC 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 ␥hENaC
Summary
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. 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. There is convincing evidence that steroids increase expression of sgk, which can in turn phosphorylate Nedd and by so doing reduce the strength of its interaction with the PY motif of ENaC [4, 5, 7, 8] This evidence supports the idea that steroid hormones and Liddle’s mutations share a common mechanism of activating ENaC. Expression of the Liddle’s mutations produced more Naϩ transport than did wild type ␥ENaC, a result that further supports the role of these mutations in producing hypertension. We found that only very small amounts of ␥ENaC protein were sufficient to produce these effects and that further increasing protein expression did not increase ENaC activity
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