A single point mutation in the TRPC3 lipid-recognition window generates supersensitivity to benzimidazole channel activators.

Mutation of a single residue within the recently identified lipid (diacylglycerol) recognition window of TRPC3 (G652A) was found to abolish channel activation via endogenous lipid mediators while retaining sensitivity to the non-lipid activator GSK1702934A (abb. GSK). The mechanism of this change in chemical sensing by TRPC3 was analysed by whole-cell and single channel electrophysiology as well as Ca imaging.

Na /Ca exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca via ER nanojunctions in vascular endothelial cells.

We investigated the role of Na/ Ca exchange (NCX) in the refilling of endoplasmic reticulum (ER) Ca in vascular endothelial cells under various conditions of cell stimulation and plasma membrane (PM) polarization. Better understanding of the mechanisms behind basic ER Ca content regulation is important, since current hypotheses on the possible ultimate causes of ER stress point to deterioration of the Ca transport mechanism to/from ER itself. We measured [Ca] temporal changes by Fura-2 fluorescence under experimental protocols that inhibit a host of transporters (NCX, Orai, non-selective transient receptor potential canonical (TRPC) channels, sarco/endoplasmic reticulum Ca ATPase (SERCA), Na/ K ATPase (NKA)) involved in the Ca communication between the extracellular space and the ER.

Functional impairment of endothelial cells by the antimycotic amphotericin B.

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.

Chiral hydroxylation at the mononuclear nonheme Fe(II) center of 4-(S) hydroxymandelate synthase--a structure-activity relationship analysis.

(S)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S)-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR) analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis.

Exploring the catalytic potential of the 3-His mononuclear nonheme Fe(II) center: discovery and characterization of an unprecedented maltol cleavage activity.

Mononuclear nonheme iron enzymes (MNHEs) catalyze a range of very diverse reactions in O(2) metabolism, but they share a common principle active-site organization. To investigate a putative catalytic promiscuity of these enzymatic metal centers, we studied the reactivity of the 3-His ligated metal center of diketone cleaving enzyme (Dke1) toward non-native substrates, with a focus on alternative O(2) dependent reactions. From a screening approach, which aims at eliminating steric factors by including minimal substrate-substructures, three alternative, 'non-β-dicarbonyl-cleavage' reactions are identified, among them an unprecedented oxygenation of maltol.