PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression.

BACKGROUND: The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 µM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. METHODS: Molecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. RESULTS: PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 µM of PA-6 inhibited outward IK1 at -50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 µM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 µM). CONCLUSIONS: 1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Levels of 25 cytokines in the first seven days of life in newborn infants.

BACKGROUND: Novel methods for cytokine analysis allow for the simultaneous measurement of 25 cytokines in 50 µL serum or plasma. Data on values of most of these cytokines in non-infected newborn infants are lacking. We analyzed levels of 25 cytokines in the first week of life in non-infected preterm and term infants and related them to gestational age. FINDINGS: During the first week after birth, no trend over time was found in any of the cytokines, except for IL-1Ra and IL-6 where higher values were found in the first four hours. Between 24 and 72 hrs levels of IL-1Ra, IL-2, IL-8, IL-12, IL-13, IL-15, IL-17, IFNγ, MIP-1a, MCP-1, TNFα were lower in infants born after 30-32 wks compared to infants ≥ 36 wks; levels of IL-6, IL-10, IP-10 were lower in preterm infants of both 30-32 and 33-36 weeks. No difference between groups for any of the levels was found for IL-1b, IL-2r, IL-4, IL-5, IL-7, IFNa, MIP-1b, GM-CSF, Eotaxin and RANTES. CONCLUSIONS: Levels of 25 interleukines are stable in the first week of life in non-infected infants. Infants born after 30-32 wks showed lower levels of fourteen cytokines compared to infants born after more then 36 wks. This indicates a lower stimulation or activation of Th-1 cells, monocytes and dendritic cells in these infants.