Probing the Ubiquitination-Mediated Function of Epithelial Sodium Channel in A549 Cells.

BACKGROUND: The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in the tight epithelia of the lung is regulated by proteolytic activation and ubiquitination. Pathophysiology of lung diseases is directly related to changes in one or both of these mechanisms. METHODS: In this study, we investigated the impact of ubiquitination and cathepsin-mediated proteolytic activation mechanisms on the functional regulation of ENaC in lung cancer A549 cells using the patch-clamp technique. RESULTS: Our findings suggest that inhibiting the proteasome (polyubiquitination) with MG132 improves ENaC activity, whereas altering the pH of the lysosome (monoubiquitination inhibition) with NH4Cl has no effect on ENaC activity. In A549 cells, inhibition of cathepsin B (CSTB) decreased the ENaC current, open probabilities (NPo and Po), and the number of active channels. CONCLUSION: These findings delineate novel modes of ENaC degradation and proteolytic activation of functional channels in A549 cells. Our findings indicate that both proteolytic activation and ubiquitination of ENaC significantly affect channel function and add new insights into the endogenous ENaC processing which might help to further understand the pathophysiology of the lung disease.

Capsaicin inhibits the function of α7-nicotinic acetylcholine receptors expressed in Xenopus oocytes and rat hippocampal neurons.

Capsaicin is a naturally occurring alkaloid derived from Chili peppers fruits. Using the two-electrode voltage-clamp technique in Xenopus oocyte expression system, actions of capsaicin on the functional properties of α7 subunit of the human nicotinic acetylcholine (α7 nACh) receptor were investigated. Ion currents activated by ACh (100 µM) were reversibly inhibited with an IC50 value of 8.6 µM. Inhibitory actions of capsaicin was independent of membrane potential. Furthermore, Ca2+-dependent Cl- channels expressed endogenously in oocytes were not involved in inhibitory actions of capsaicin. In addition, increasing the ACh concentrations could not reverse the inhibitory effects of capsaicin. Importantly, specific binding of [125I] α-bungarotoxin remained unaltered by capsaicin suggesting that its effect is noncompetitive. Whole cell patch-clamp technique was performed in CA1 stratum radiatum interneurons of rat hippocampal slices. Ion currents induced by choline, a selective-agonist of α7-receptor, were reversibly inhibited by 10 min bath application of capsaicin (10 µM). Collectively, results of our investigation indicate that the function of the α7-nACh receptor expressed in Xenopus oocytes and in hippocampal interneurons are inhibited by capsaicin.