Molekulare Charakterisierung des epithelialen Na+-Kanals (ENaC) aus Nasengewebe von Patienten mit und ohne Mukoviszidose

Mukoviszidose (Cystische Fibrose, CF) beruht auf einem Gendefekt im CFTR und ist charakterisiert durch eine reduzierte Cl-Sekretion und eine drastisch erhöhte Na+-Absorption, die durch ENaC hervorgerufen wird. Während viele CFTR-Mutationen untersucht und beschrieben wurden, blieben die molekularen Mechanismen, welche die Na+-Hyperabsorption durch ENaC verursachen, weitgehend unaufgeklärt. Um Mutationen im ENaC ausschließen zu können, die eine veränderte Kanaleigenschaft hervorrufen würden und somit zu einer gesteigerten Na+-Absorption führen könnten, wurde der humane ENaC aus respiratorischem Nasenepithel (hnENaC) von CF und nicht-CF Patienten kloniert und sequenziert. So konnte deutlich gezeigt werden, dass die Sequenzen des hnENaC von CF-Patienten und nicht-CF Patienten identisch sind. Nachgewiesen werden konnte darüber hinaus, dass die Menge an ENaC im Nasenepithel von CF Patienten auf mRNA- und teilweise auch Protein-Ebene im direkten Vergleich mit den nicht-CF Proben erhöht ist

Upregulated expression of ENaC in human CF nasal epithelium

AbstractCystic fibrosis (CF) is characterised by the absence of CFTR function resulting in a reduced Cl− secretion and an increase in Na+ absorption. This Na+ hyperabsorption is mediated by the human amiloride-sensitive epithelial sodium channel (ENaC), but the underlying mechanisms are still unknown. After demonstrating functional differences of the Na+ absorption in CF and non-CF epithelia in Ussing chamber experiments with human primary cultures, we compared ENaC sequences from CF and non-CF human nasal tissue (hnENaC), investigated the mRNA transcription levels via real-time PCR and studied the protein expression in Western blot analyses. We found no differences in the sequences of CF and non-CF hnENaC, but identified some polymorphisms. The real-time experiments revealed an enhanced mRNA amount of all three hnENaC subunits in CF tissue. By comparing the two groups on the protein level, we observed differences in the abundance of the Na+ channel. While the α- and β-hnENaC protein amount was increased in CF tissue the γ-hnENaC was decreased. We conclude that the Na+ hyperabsorption in CF is not caused by mutations in hnENaC, but by an increase in the transcription of the hnENaC subunits. This could be induced by a disturbed regulation of the channel in CF

Expression of ENaC and other Transport Proteins in Xenopus Oocytes is Modulated by Intracellular Na+

The expression of the epithelial Na+ channel (ENaC) is tissue-specific and dependent on a variety of mediators and interacting proteins. Here we examined the role of intracellular Na+ ([Na+](i)) as a modulator of the expression of rat ENaC in Xenopus laevis oocytes. We manipulated [Na+](i) of ENaC-expressing oocytes in the range of 0-20 mM by incubating in extracellular solutions of different [Na+](o). Electrophysiological, protein biochemical and fluorescence optical methods were used to determine the effects of different [Na+]i on ENaC expression and membrane abundance. In voltage-clamp experiments we found that amiloride-sensitive ENaC current (Iami) and conductance (Gami) peak at a [Na+](i) of approximately 10 mM Na+, but were significantly reduced in 5 mM and 20 mM [Na+](i). Fluorescence intensity of EGFP-ENaC-expressing oocytes also followed a bell-shaped curve with a maximum at approximately 10 mM [Na+](i). In Western blot experiments with specific anti-ENaC antibodies the highest protein expression was found in ENaC-expressing oocytes with [Na+](i) of 10-15 mM. Since ENaC is also highly permeable for Li+, we incubated ENaC-expressing oocytes in different Li+ concentrations and found a peak of Iami and Gami with 5 mM Li+. The influence of [Na+](i) on the expression is not ENaC-specific, since expression of a Cl(-) channel (CFTR) and a Na+/glucose cotransporter (SGLT1) showed the same dependence on [Na+](i). We conclude that specific concentrations of Na+ and Li+ influence the expression and abundance of ENaC and other transport proteins in the plasma membrane in Xenopus laevis oocytes. Furthermore, we suggest the existence of a general mechanism dependent on monovalent cations that optimizes the expression of membrane proteins.status: publishe

Acute effects of deep brain stimulation on brain function in obsessive-compulsive disorder

OBJECTIVE: Deep brain stimulation (DBS) is an effective treatment for refractory obsessive-compulsive disorder (OCD) yet neural markers of optimized stimulation parameters are largely unknown. We aimed to describe (sub-)cortical electrophysiological responses to acute DBS at various voltages in OCD. METHODS: We explored how DBS doses between 3-5 V delivered to the ventral anterior limb of the internal capsule of five OCD patients affected electroencephalograms and intracranial local field potentials (LFPs). We focused on theta power/ phase-stability, given their previously established role in DBS for OCD. RESULTS: Cortical theta power and theta phase-stability did not increase significantly with DBS voltage. DBS-induced theta power peaks were seen at the previously defined individualized therapeutic voltage. Although LFP power generally increased with DBS voltages, this occurred mostly in frequency peaks that overlapped with stimulation artifacts limiting its interpretability. Though highly idiosyncratic, three subjects showed significant acute DBS effects on electroencephalogram theta power and four subjects showed significant carry-over effects (pre-vs post DBS, unstimulated) on LFP and electroencephalogram theta power. CONCLUSIONS: Our findings challenge the presence of a consistent dose-response relationship between stimulation voltage and brain activity. SIGNIFICANCE: Theta power may be investigated further as a neurophysiological marker to aid personalized DBS voltage optimization in OCD