IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.