Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels

KCNQ1 (Kv7.1), together with its KCNE β subunits, plays a pivotal role both in the repolarization of cardiac tissue and in water and salt transport across epithelial membranes. Nedd4/Nedd4-like (neuronal precursor cell-expressed developmentally downregulated 4) ubiquitin-protein ligases interact with the KCNQ1 potassium channel through a PY motif located in the C terminus of KCNQ1. This interaction induces ubiquitylation of KCNQ1, resulting in a reduced surface density of the channel. It was reported recently that the epithelial sodium channel is regulated by the reverse process-deubiquitylation-mediated by USP2 (ubiquitin-specific protease 2).

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Stereoselective Inhibition of the hERG1 Potassium Channel

A growing number of drugs have been shown to prolong cardiac repolarization, predisposing individuals to life-threatening ventricular arrhythmias known as Torsades de Pointes. Most of these drugs are known to interfere with the human ether à-gogo related gene 1 (hERG1) channel, whose current is one of the main determinants of action potential duration. Prolonged repolarization is reflected by lengthening of the QT interval of the electrocardiogram, as seen in the suitably named drug-induced long QT syndrome. Chirality (presence of an asymmetric atom) is a common feature of marketed drugs, which can therefore exist in at least two enantiomers with distinct three-dimensional structures and possibly distinct biological fates. Both the pharmacokinetic and pharmacodynamic properties can differ between enantiomers, as well as also between individuals who take the drug due to metabolic polymorphisms. Despite the large number of reports about drugs reducing the hERG1 current, potential stereoselective contributions have only been scarcely investigated. In this review, we present a non-exhaustive list of clinically important molecules which display chiral toxicity that may be related to hERG1-blocking properties. We particularly focus on methadone cardiotoxicity, which illustrates the importance of the stereoselective effect of drug chirality as well as individual variations resulting from pharmacogenetics. Furthermore, it seems likely that, during drug development, consideration of chirality in lead optimization and systematic assessment of the hERG1 current block with all enantiomers could contribute to the reduction of the risk of drug-induced LQTS.

Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate

The aim of this study was to evaluate the 4-year clinical outcomes following regenerative surgery in intrabony defects with either EMD + BCP or EMD. Twenty-four patients with advanced chronic periodontitis, displaying one-, two-, or three-walled intrabony defect with a probing depth of at least 6 mm, were randomly treated with either EMD + BCP (test) or EMD alone (control). The following clinical parameters were evaluated at baseline, at 1 year and at 4 years after regenerative surgery: plaque index, gingival index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. No differences in any of the investigated parameters were observed at baseline between the two groups. The test group demonstrated a mean CAL change from from 10.8 ± 1.6 mm to 7.4 ± 1.6 mm (p < 0.001) and to 7.6 ± 1.7 mm (p < 0.001) at 1 and 4 years, respectively. In the control group, mean CAL changed from 10.4 ± 1.3 at baseline to 6.9 ± 1.0 mm (p < 0.001) at 1 year and 7.2 ± 1.2 mm (p < 0.001) at 4 years. At 4 years, two defects in the test group and three defects in the control group have lost 1 mm of the CAL gained at 1 year. Compared to baseline, at 4 years, a CAL gain of ≥3 mm was measured in 67% of the defects (i.e., in 8 out of 12) in the test group and in 75% of the defects (i.e., in 9 out of 12) in the control group. There were no statistically significant differences in any of the investigated parameters at 1 and at 4 years between the two groups. Within their limits, the present results indicate that: (a) the clinical improvements obtained with both treatments can be maintained over a period of 4 years, and (b) in two- and three-walled intrabony defects, the addition of BCP did not additionally improve the outcomes obtained with EMD alone. In two- and three-walled intrabony defects, the combination of EMD + BCP did not show any advantage over the use of EMD alone.

Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice

Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown.

The immunomodulator FTY720 and its phosphorylated derivative activate the Smad signalling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells

1.--The immunomodulating agent FTY720 is a substrate for the sphingosine kinase and the phosphorylated form is able to bind to sphingosine 1-phosphate (S1P) receptors. In this study, we show that exposure of renal mesangial cells to phospho-FTY720 leads to a rapid and transient activation of several protein kinase cascades, including the mitogen- and stress-activated protein kinases. The nonphosphorylated FTY720 also increased MAPK phosphorylation, but with a reduced potency and a more delayed time course. In addition, phospho-FTY720 and FTY720 are able to increase phosphorylation of Smad proteins which are classical members of the transforming growth factor-beta (TGF-beta) signalling device, thus suggesting a crosstalk between FTY720 and TGF-beta signalling. 2.--Pretreatment with the S1P(3) receptor antagonist suramin inhibits FTY720 and phospho-FTY720-induced Smad phosphorylation, whereas pertussis toxin pretreatment, which blocks G(i/0) proteins, has no effect on Smad phosphorylation. 3.--Since TGF-beta is a potent profibrotic cytokine in mesangial cells and upregulates the connective tissue growth factor (CTGF) and collagen as important hallmarks in the fibrotic sequelae, we investigated whether FTY720 and phospho-FTY720 are able to mimic these effects of TGF-beta. Indeed, FTY720 and phospho-FTY720 markedly upregulate CTGF and collagen type IV protein expressions. In addition, the tissue inhibitor of metalloproteinase-1 is transcriptionally activated by FTY720, whereas cytokine-induced matrix metalloproteinase-9 is down-regulated by FTY720. 4.--Depletion of the TGF-beta receptor type II by the siRNA transfection technique blocks not only Smad phosphorylation but also CTGF upregulation. Similarly, Smad-4 depletion by siRNA transfection also abrogates CTGF upregulation induced by FTY720 and phospho-FTY720. 5.--In summary, our data show that FTY720 and phospho-FTY720 not only activate the Smad signalling cascade in mesangial cells, but also upregulate the expression of CTGF and collagen. These findings suggest that FTY720 may have additional effects besides the established immunomodulatory action and, importantly, a profibrotic activity has to be considered in future experimental approaches.

DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours

PURPOSE: We aimed at designing and developing a novel bombesin analogue, DOTA-PEG(4)-BN(7-14) (DOTA-PESIN), with the goal of labelling it with (67/68)Ga and (177)Lu for diagnosis and radionuclide therapy of prostate and other human cancers overexpressing bombesin receptors. METHODS: The 8-amino acid peptide bombesin (7-14) was coupled to the macrocyclic chelator DOTA via the spacer 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG(4)). The conjugate was complexed with Ga(III) and Lu(III) salts. The GRP receptor affinity and the bombesin receptor subtype profile were determined in human tumour specimens expressing the three bombesin receptor subtypes. Internalisation and efflux studies were performed with the human GRP receptor cell line PC-3. Xenografted nude mice were used for biodistribution. RESULTS: [Ga(III)/Lu(III)]-DOTA-PESIN showed good affinity to GRP and neuromedin B receptors but no affinity to BB3. [(67)Ga/(177)Lu]-DOTA-PESIN internalised rapidly into PC-3 cells whereas the efflux from PC-3 cells was relatively slow. In vivo experiments showed a high and specific tumour uptake and good retention of [(67)Ga/(177)Lu]-DOTA-PESIN. [(67)Ga/(177)Lu]-DOTA-PESIN highly accumulated in GRP receptor-expressing mouse pancreas. The uptake specificity was demonstrated by blocking tumour uptake and pancreas uptake. Fast clearance was found from blood and all non-target organs except the kidneys. High tumour-to-normal tissue ratios were achieved, which increased with time. PET imaging with [(68)Ga]-DOTA-PESIN was successful in visualising the tumour at 1 h post injection. Planar scintigraphic imaging showed that the (177)Lu-labelled peptide remained in the tumour even 3 days post injection. CONCLUSION: The newly designed ligands have high potential with regard to PET and SPECT imaging with (68/67)Ga and targeted radionuclide therapy with (177)Lu.

Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous deletion/inactivation of the survival of motoneuron 1 (SMN1) gene. The nearby SMN2 gene, despite its identical coding capacity, is only an incomplete substitute, because a single nucleotide difference impairs the inclusion of its seventh exon in the messenger RNA (mRNA). This splicing defect can be corrected (transiently) by specially designed oligonucleotides. Here we have developed a more permanent correction strategy based on bifunctional U7 small nuclear RNAs (snRNAs). These carry both an antisense sequence that allows specific binding to exon 7 and a splicing enhancer sequence that will improve the recognition of the targeted exon. When expression cassettes for these RNAs are stably introduced into cells, the U7 snRNAs become incorporated into small nuclear ribonucleoprotein (snRNP) particles that will induce a durable splicing correction. We have optimized this strategy to the point that virtually all SMN2 pre-mRNA becomes correctly spliced. In fibroblasts from an SMA patient, this approach induces a prolonged restoration of SMN protein and ensures its correct localization to discrete nuclear foci (gems).

Potassium transporters in plants--involvement in K+ acquisition, redistribution and homeostasis

Potassium is a major plant nutrient which has to be accumulated in great quantity by roots and distributed throughout the plant and within plant cells. Membrane transport of potassium can be mediated by potassium channels and secondary potassium transporters. Plant potassium transporters are present in three families of membrane proteins: the K(+) uptake permeases (KT/HAK/KUP), the K(+) transporter (Trk/HKT) family and the cation proton antiporters (CPA). This review will discuss the contribution of members of each family to potassium acquisition, redistribution and homeostasis.

High level of extracellular potassium and its correlates after severe head injury: relationship to high intracranial pressure

OBJECT: Disturbed ionic and neurotransmitter homeostasis are now recognized as probably the most important mechanisms contributing to the development of secondary brain swelling after traumatic brain injury (TBI). Evidence obtained in animal models indicates that posttraumatic neuronal excitation by excitatory amino acids leads to an increase in extracellular potassium, probably due to ion channel activation. The purpose of this study was therefore to measure dialysate potassium in severely head injured patients and to correlate these results with measurements of intracranial pressure (ICP), patient outcome, and levels of dialysate glutamate and lactate, and cerebral blood flow (CBF) to determine the role of ischemia in this posttraumatic ion dysfunction. METHODS: Eighty-five patients with severe TBI (Glasgow Coma Scale Score < 8) were treated according to an intensive ICP management-focused protocol. All patients underwent intracerebral microdialyis. Dialysate potassium levels were analyzed using flame photometry, and dialysate glutamate and dialysate lactate levels were measured using high-performance liquid chromatography and an enzyme-linked amperometric method in 72 and 84 patients, respectively. Cerebral blood flow studies (stable xenon computerized tomography scanning) were performed in 59 patients. In approximately 20% of the patients, dialysate potassium values were increased (dialysate potassium > 1.8 mM) for 3 hours or more. A mean amount of dialysate potassium greater than 2 mM throughout the entire monitoring period was associated with ICP above 30 mm Hg and fatal outcome, as were progressively rising levels of dialysate potassium. The presence of dialysate potassium correlated positively with dialysate glutamate (p < 0.0001) and lactate (p < 0.0001) levels. Dialysate potassium was significantly inversely correlated with reduced CBF (p = 0.019). CONCLUSIONS: Dialysate potassium was increased after TBI in 20% of measurements. High levels of dialysate potassium were associated with increased ICP and poor outcome. The simultaneous increase in dialysate potassium, together with dialysate glutamate and lactate, supports the concept that glutamate induces ionic flux and consequently increases ICP, which the authors speculate may be due to astrocytic swelling. Reduced CBF was also significantly correlated with increased levels of dialysate potassium. This may be due to either cell swelling or altered vasoreactivity in cerebral blood vessels caused by higher levels of potassium after trauma. Additional studies in which potassium-sensitive microelectrodes are used are needed to validate these ionic events more clearly.

High extracellular potassium and its correlates after severe head injury: relationship to high intracranial pressure

Disturbed ionic and neurotransmitter homeostasis are now recognized to be probably the most important mechanisms contributing to the development of secondary brain swelling after traumatic brian injury (TBI). Evidence obtained from animal models indicates that posttraumatic neuronal excitation via excitatory amino acids leads to an increase in extracellular potassium, probably due to ion channel activation. The purpose of this study was therefore to measure dialysate potassium in severely head injured patients and to correlate these results with intracranial pressure (ICP), outcome, and also with the levels of dialysate glutamate, lactate, and cerebral blood flow (CBF) so as to determine the role of ischemia in this posttraumatic ionic dysfunction. Eighty-five patients with severe TBI (Glasgow Coma Scale score < 8) were treated according to an intensive ICP management-focused protocol. All patients underwent intracerebral microdialyis. Dialysate potassium levels were analyzed by flame photometry, as were dialysate glutamate and dialysate lactate levels, which were measured using high-performance liquid chromatography and an enzyme-linked amperometric method in 72 and 84 patients respectively. Cerebral blood flow studies (stable Xenon--computerized tomography scanning) were performed in 59 patients. In approximately 20% of the patients, potassium values were increased (dialysate potassium > 1.8 mmol). Mean dialysate potassium (> 2 mmol) was associated with ICP above 30 mm Hg and fatal outcome. Dialysate potassium correlated positively with dialysate glutamate (p < 0.0001) and lactate levels (p < 0.0001). Dialysate potassium was significantly inversely correlated with reduced CBF (p = 0.019). Dialysate potassium was increased after TBI in 20% of measurements. High levels of dialysate potassium were associated with increased ICP and poor outcome. The simultaneous increase of potassium, together with dialysate glutamate and lactate, supports the hypothesis that glutamate induces ionic flux and consequently increases ICP due to astrocytic swelling. Reduced CBF was also significantly correlated with increased levels of dialysate potassium. This may be due to either cell swelling or altered potassium reactivity in cerebral blood vessels after trauma.