The normalized slope conductance as a tool for quantitative analysis of current-voltage relations.

The patch-clamp method, which was awarded the Nobel Prize in 1991, is a well-established and indispensable method to study ion channels in living cells and to biophysically characterize non-voltage-gated ion channels, which comprise about 70% of all ion channels in the human genome. To investigate the biophysical properties of non-voltage-gated ion channels, whole-cell measurements with application of continuous voltage ramps are routinely conducted to obtain current-voltage (IV) relationships. However, adequate tools for detailed and quantitative analysis of IV curves are still missing. We use the example of the transient receptor potential classical (TRPC) channel family to elucidate whether the normalized slope conductance (NSC) is an appropriate tool for reliable discrimination of the IV curves of diverse TRPC channels that differ in their individual curve progression. We provide a robust calculation method for the NSC, and, by applying this method, we find that TRPC channel activators and modulators can evoke different NSC progressions independent from their expression levels, which points to distinguishable active channel states. TRPC6 mutations in patients with focal segmental glomerulosclerosis resulted in distinct NSC progressions, suggesting that the NSC is suitable for investigating structure-function relations and might help unravel the unknown pathomechanisms leading to focal segmental glomerulosclerosis. The NSC is an effective algorithm for extended biophysical characterization of non-voltage-gated ion channels.

Shape Similarity by Fractal Dimensionality: An Application in the de novo Design of (-)-Englerin A Mimetics.

Molecular shape and pharmacological function are interconnected. To capture shape, the fractal dimensionality concept was employed, providing a natural similarity measure for the virtual screening of de novo generated small molecules mimicking the structurally complex natural product (-)-englerin A. Two of the top-ranking designs were synthesized and tested for their ability to modulate transient receptor potential (TRP) cation channels which are cellular targets of (-)-englerin A. Intracellular calcium assays and electrophysiological whole-cell measurements of TRPC4 and TRPM8 channels revealed potent inhibitory effects of one of the computer-generated compounds. Four derivatives of this identified hit compound had comparable effects on TRPC4 and TRPM8. The results of this study corroborate the use of fractal dimensionality as an innovative shape-based molecular representation for molecular scaffold-hopping.

The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans.

UNLABELLED: The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans Under these conditions, both microorganisms synthesized pseudo-B12 This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-(13)C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. IMPORTANCE: The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes. Selected cobamide-containing enzymes (e.g., reductive dehalogenases) showed specificity for their cobamide cofactors. It has recently been proposed that the structure of the linker moiety of norpseudo-B12 and the mode of binding of the EA-P linker to the PceA enzyme reflect the high specificity of the enzyme for its cofactor. Data reported herein do not support this idea. In fact, norpseudo-B12 was functional in the cobamide-dependent methionine biosynthesis of S. enterica, raising questions about the role of norcobamides in nature.