A high affinity switch for cAMP in the HCN pacemaker channels.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate electrical rhythmicity in special brain neurons and cardiomyocytes. The channels are activated by membrane hyperpolarization. The binding of cAMP to the four available cyclic nucleotide-binding domains (CNBD) enhances channel activation. We analyzed in the present study the mechanism of how the effect of cAMP binding is transmitted to the pore domain. Our strategy was to uncouple the C-linker (CL) from the channel core by inserting one to five glycine residues between the S6 gate and the A'-helix (constructs 1G to 5G). We quantified in full-length HCN2 channels the resulting functional effects of the inserted glycines by current activation as well as the structural dynamics and statics using molecular dynamics simulations and Constraint Network Analysis. We show functionally that already in 1G the cAMP effect on activation is lost and that with the exception of 3G and 5G the concentration-activation relationships are shifted to depolarized voltages with respect to HCN2. The strongest effect was found for 4G. Accordingly, the activation kinetics were accelerated by all constructs, again with the strongest effect in 4G. The simulations reveal that the average residue mobility of the CL and CNBD domains is increased in all constructs and that the junction between the S6 and A'-helix is turned into a flexible hinge, resulting in a destabilized gate in all constructs. Moreover, for 3G and 4G, there is a stronger downward displacement of the CL-CNBD than in HCN2 and the other constructs, resulting in an increased kink angle between S6 and A'-helix, which in turn loosens contacts between the S4-helix and the CL. This is suggested to promote a downward movement of the S4-helix, similar to the effect of hyperpolarization. In addition, exclusively in 4G, the selectivity filter in the upper pore region and parts of the S4-helix are destabilized. The results provide new insights into the intricate activation of HCN2 channels.

Novel Fluorescent Cyclic Nucleotide Derivatives to Study CNG and HCN Channel Function.

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.

Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy - entropy compensation.

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding.

Placental immune response to apple allergen in allergic mothers.

The immunological milieu in the placenta may be crucial for priming the developing foetal immune system. Early imbalances may promote the establishment of immune-mediated diseases in later life, including allergies. The initial exposure to allergens seems to occur in utero, but little is known about allergen-induced placental cytokine and chemokine release. The release of several cytokines and chemokines from placenta tissue after exposure to mast cell degranulator compound 48/80 or apple allergen in placentas from allergic and healthy mothers was to be analysed. Four placentas from women with apple allergy and three controls were applied in a placental perfusion model with two separate cotyledons simultaneously perfused with and without apple allergen (Mal d 1). Two control placentas were perfused with compound 48/80. In outflow, histamine was quantified spectrophotofluorometrically, IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ by a cytometric multiplex bead array and IL-13 and CXCL10, CXCL11, CCL17 and CCL22 with an in-house multiplex Luminex assay. Compound 48/80 induced a rapid release of histamine, CXCL10, CXCL11, CCL17 and CCL22, but not of the other factors. Apple allergen induced a time-dependent release of IL-6 and TNF, but not of histamine, in placentas of women with apple allergy compared with the unstimulated cotyledon. CCL17 levels were slightly increased after allergen stimulation in control placentas. Allergens can induce placental cytokines and chemokines distinctly in allergic and healthy mothers. These mediators may affect the prenatal development of the immune system and modify the risk of diseases related to immune disorders in childhood such as allergies.

Phthalate exposure in pregnant women and newborns - the urinary metabolite excretion pattern differs distinctly.

Some phthalates are endocrine disruptors and reproductive and developmental toxicants. Data on newborn phthalate exposure and elimination characteristics are scarce. We determined 21 urinary phthalate metabolites (indicating exposure to 11 parent phthalates) in two study approaches: in the first approach we collected the urine of 20 healthy newborns at days 2-5 post partum together with 47 urine samples of 7 women during pregnancy. In the second fine tuned approach we collected first urine samples of 9 healthy newborns together with their mother's urine shortly before birth. To ensure full and contamination free collection of the newborns first urines we used special adhesive urine bags for children. All urine samples revealed ubiquitous exposures to phthalates comparable to other populations. Metabolite levels in the newborns first day urine samples were generally lower than in all other samples. However, the newborns urines (both first and day 2-5 urines) showed a metabolite pattern distinctly different from the maternal and general population samples: in the newborns urines the carboxy-metabolites of the long chain phthalates (DEHP, DiNP, DiDP) were the by far dominant metabolites with a relative share in the metabolite spectrum up to 6 times higher than in maternal urine. Oppositely, for the short chain phthalates (DBP, DiBP) oxidized metabolites seemed to be less favored than the simple monoesters in the newborns urines. The skewed metabolite distribution in the newborns urine warrants further investigation in terms of early phthalate metabolism, the quantity of internal phthalate exposure of the fetus/newborn and its possible health effects.

Fatty acid distribution of cord and maternal blood in human pregnancy: special focus on individual trans fatty acids and conjugated linoleic acids.

BACKGROUND: Maternal nutrition in pregnancy has a crucial impact on the development of the fetus. Dietary trans fatty acids (tFA) are known to have adverse health effects, especially during pregnancy. However, the distribution of tFA produced via partial hydrogenation of vegetable oils (mainly elaidic acid; t9) differs compared to ruminant-derived tFA (mainly vaccenic acid; t11). Recent findings indicate that they may have different impact on human health.Therefore, in this study, plasma and erythrocytes of mother-child pairs (n = 55) were sampled to investigate the distribution of tFA, including individual trans C18:1 fatty acids and conjugated linoleic acids (CLA) in fetal related to maternal lipids; with additional consideration of maternal dairy fat intake. RESULTS: Portion of t9 and t11, but also of c9,t11 CLA was higher in maternal than in fetal blood lipids. The portion of t9 in maternal and fetal lipids differed only slightly. In contrast, the portion of fetal t11 was only half of that in maternal blood. This led to a fetal t9/t11-index in plasma and erythrocytes being twice as high compared to the maternal values. A high dairy fat intake resulted in elevated portions of t11 and its Δ9-desaturation product c9,t11 CLA in maternal blood. In contrast, in the respective fetal blood lipids only c9,t11 CLA, but not t11 was increased. Nevertheless, a positive association between maternal and fetal plasma exists for both t11 and c9,t11 CLA. Furthermore, in contrast to t9, t11 was not negatively associated with n-3 LC-PUFA in fetal blood lipids. CONCLUSIONS: Fetal blood fatty acid composition essentially depends on and is altered by the maternal fatty acid supply. However, in addition to dietary factors, other aspects also contribute to the individual fatty acid distribution (oxidation, conversion, incorporation). The lower portion of fetal t11 compared to maternal t11, possibly results from Δ9-desaturation to c9,t11 CLA and/or oxidation. Based on the fatty acid distribution, it can be concluded that t11 differs from t9 regarding its metabolism and their impact on fetal LC-PUFA.

Maternal diet during pregnancy has tissue-specific effects upon fetal fatty acid composition and alters fetal immune parameters.

Both animal and human studies demonstrate that the docosahexaenoic acid (DHA) content of plasma and/or tissue lipids is increased during pregnancy. We hypothesised that increasing the α-linolenic acid (ALA) or longer chain (n-3) PUFA content of the maternal diet during pregnancy influences fetal fatty acid composition and the fetal immune system. Pregnant rats were fed a low-fat (LF) soybean oil diet, or high-fat (HF) soybean, linseed, salmon or sunflower oil diets from conception to 20d gestation. The ALA-rich Linseed-HF diet resulted in an equivalent eicosapentaenoic acid (EPA) status in fetal immune tissues and an equivalent DHA status in the fetal brain to that achieved with the Salmon-HF diet. An (n-3) rich maternal diet during pregnancy associated with the highest expression of CD3 (Salmon-HF) and CD8 (Linseed-HF and Salmon-HF) on fetal thymic CD3(+)CD8(+) cells. The Linseed-HF diet resulted in the highest proportion of CD161(+) cells within the fetal thymus, which correlated with the production of IL-4. These data indicate that dietary ALA supplementation may confer some of the benefits of LC (n-3) PUFA during pregnancy. This should be examined in suitably designed human studies.

Development of a human model to study homing behavior of immune cells into decidua and placental villi under ex vivo conditions.

PROBLEM: Homing of lymphocytes and NK cells into the decidua and its regulation has been very controversially discussed. Therefore, we aimed to establish an in vivo simulation method for analysis of homing behavior, which might be also useful for other cells such as stem or tumor cells. METHOD OF STUDY: A human term placenta has been perfused with medium to elute blood and then with maternal autologous carboxyfluorescein succinimidyl ester (CFSE)-labeled peripheral blood lymphocytes for 3 hr and rinsed for another 2 hr. Tissue was analysed histologically for detection of labeled cells. Labeled lymphocytes and beads in perfusate have been identified and counted by flow cytometry. RESULTS: At the moment of tissue fixation for histology, the perfusate was free of labeled cells. Labeled perfused lymphocytes have been found adhered and integrated in vessel wall structures, in decidual stroma and as colonies in individual villi. CONCLUSION: Placenta perfusion with a lymphocyte suspension is feasible without plugging the tube system. Time is sufficient for cells to adhere and to migrate into the stroma. Also some villi have been infiltrated which might be caused by inflammatory stimuli. The perfusion system might be useful to test substances for their capacity to influence homing of lymphocytes or other cells.

Impact of PUFA on early immune and fetal development.

It has recently been reported that the increased prevalence in childhood allergy may be linked to deviations in fetal immune development. One reason may be impaired nutrient supply. Hence, a well-differentiated placenta together with an optimal fetal nutrition via the mother are important prerequisites for the establishment of a functional immune system with normal immune responses. Fatty acids and their derivatives can influence both the early immune development and immune maturation by regulating numerous metabolic processes and the gene expression of important proteins such as enzymes and cytokines. The present review summarises the impact of nutritional fatty acids on the development of the immune system as well as the fetal development. It describes the mechanisms of action of PUFA, trans fatty acids and conjugated linoleic acids in programming the fetus with regard to its risk of acquiring atopic diseases in childhood.