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Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome
1Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany.
Abstract:
Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.
Insights
Cystic fibrosis transmembrane conductance regulator (CFTR) inhibits epithelial sodium channels (ENaC) independently of Nedd4-mediated ubiquitination. This study reveals CFTR
Area of Science:
- Physiology
- Molecular Biology
- Ion Channel Function
Background:
- Epithelial Na+ channels (ENaC) are crucial for sodium absorption.
- The cystic fibrosis transmembrane conductance regulator (CFTR) is known to inhibit ENaC activity.
- The precise mechanism by which CFTR regulates ENaC remains incompletely understood, particularly regarding endocytosis pathways.
Purpose of the Study:
- To investigate whether CFTR regulates ENaC by interfering with Nedd4- and ubiquitin-mediated endocytosis.
- To elucidate the molecular mechanisms underlying CFTR's inhibitory effect on ENaC.
- To determine if CFTR-dependent regulation of ENaC is independent of ubiquitination pathways.
Main Methods:
- Introduction of C-terminal mutations in alpha-, beta-, and gamma-subunits of rat ENaC to eliminate PY motifs, crucial for Nedd4 binding.
- Expression of wild-type and mutant ENaC subunits in Xenopus oocytes.
- Measurement of whole-cell Na+ currents using electrophysiology.
- Assessment of CFTR-dependent inhibition using forskolin and IBMX.
- Utilizing antibodies against dynamin and ubiquitin to probe endocytosis pathways.
Main Results:
- Mutations eliminating PY motifs in ENaC subunits resulted in enhanced Na+ currents compared to wild-type.
- Despite enhanced currents, ENaC activity in all mutants was inhibited by activated CFTR.
- Inhibition of ENaC by CFTR was not affected by antibodies to dynamin or ubiquitin.
- CFTR-dependent inhibition of ENaC was observed even when ENaC activity was modulated by proteases or xCAP1.
Conclusions:
- CFTR regulation of ENaC activity is independent of Nedd4-dependent ubiquitination and endocytosis.
- CFTR's inhibitory mechanism on ENaC operates downstream of ubiquitination and endocytic pathways.
- The findings suggest a distinct regulatory pathway for CFTR's control over ENaC function.