An inhibitory peptide derived from the α-subunit of the epithelial sodium channel (ENaC) shows a helical conformation.

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αßγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αßγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.

Complementary use of 1H NMR and multi-element IRMS in association with chemometrics enables effective origin analysis of cocoa beans (Theobroma cacao L.).

Within the cocoa market (Theobroma cacao L.), quality and prices are often determined by geographical origin, making traceability indispensable. Therefore, to investigate possibilities of tracing by analytical methods, 48 carefully selected cocoa samples from 20 countries have been profiled using a combination of stable isotope-ratio mass spectrometry (IRMS) and proton nuclear magnetic resonance (1H NMR). Chemometric analysis of combined data sets from both, stable isotope data (δ13C, δ15N, δ18O, δ2H, %C, %N, %O, %H) and 1H NMR fingerprints, achieved good separation with increased classification rates compared to classification with data of the isolated methods. IRMS contributed primarily to discrimination between countries, while 1H NMR significantly contributed to separation of varieties, but also the regions within individual countries. This study thus demonstrates that combination of two analytical methods is an effective tool to enhance both, accuracy and precision, in authenticity testing of cocoa.