Loss-of-function mutation of the SCN3B-encoded sodium channel {beta}3 subunit associated with a case of idiopathic ventricular fibrillation.

AIMS Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. METHODS AND RESULTS Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. CONCLUSION This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.

OBJECTIVES This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). BACKGROUND Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. METHODS Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 +/- 15 years, mean QTc 428 +/- 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). RESULTS Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. CONCLUSIONS Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that approximately 65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

SCN4B-encoded sodium channel beta4 subunit in congenital long-QT syndrome.

BACKGROUND Congenital long-QT syndrome (LQTS) is potentially lethal secondary to malignant ventricular arrhythmias and is caused predominantly by mutations in genes that encode cardiac ion channels. Nearly 25% of patients remain without a genetic diagnosis, and genes that encode cardiac channel regulatory proteins represent attractive candidates. Voltage-gated sodium channels have a pore-forming alpha-subunit associated with 1 or more auxiliary beta-subunits. Four different beta-subunits have been described. All are detectable in cardiac tissue, but none have yet been linked to any heritable arrhythmia syndrome. METHODS AND RESULTS We present a case of a 21-month-old Mexican-mestizo female with intermittent 2:1 atrioventricular block and a corrected QT interval of 712 ms. Comprehensive open reading frame/splice mutational analysis of the 9 established LQTS-susceptibility genes proved negative, and complete mutational analysis of the 4 Na(vbeta)-subunits revealed a L179F (C535T) missense mutation in SCN4B that cosegregated properly throughout a 3-generation pedigree and was absent in 800 reference alleles. After this discovery, SCN4B was analyzed in 262 genotype-negative LQTS patients (96% white), but no further mutations were found. L179F was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells that contained the stably expressed SCN5A-encoded sodium channel alpha-subunit (hNa(V)1.5). Compared with the wild-type, L179F-beta4 caused an 8-fold (compared with SCN5A alone) and 3-fold (compared with SCN5A + WT-beta4) increase in late sodium current consistent with the molecular/electrophysiological phenotype previously shown for LQTS-associated mutations. CONCLUSIONS We provide the seminal report of SCN4B-encoded Na(vbeta)4 as a novel LQT3-susceptibility gene.

The channel-activating protease CAP1/Prss8 is required for placental labyrinth maturation

The serine protease CAP1/Prss8 is crucial for skin barrier function, lung alveolar fluid clearance and has been unveiled as diagnostic marker for specific cancer types. Here, we show that a constitutive knockout of CAP1/Prss8 leads to embryonic lethality. These embryos presented no specific defects, but it is during this period, and in particular at E13.5, that wildtype placentas show an increased expression of CAP1/Prss8, thus suggesting a placental defect in the knockout situation. The placentas of knockout embryos exhibited significantly reduced vascular development and incomplete cellular maturation. In contrary, epiblast-specific deletion of CAP1/Prss8 allowed development until birth. These CAP1/Prss8-deficient newborns presented abnormal epidermis, and died soon after birth due to impaired skin function. We thus conclude that a late placental insufficiency might be the primary cause of embryonic lethality in CAP1/Prss8 knockouts. This study highlights a novel and crucial role for CAP1/Prss8 in placental development and function.

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.

Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel.

Brugada syndrome (BrS) is a condition defined by ST-segment alteration in right precordial leads and a risk of sudden death. Because BrS is often associated with right bundle branch block and the TRPM4 gene is involved in conduction blocks, we screened TRPM4 for anomalies in BrS cases. The DNA of 248 BrS cases with no SCN5A mutations were screened for TRPM4 mutations. Among this cohort, 20 patients had 11 TRPM4 mutations. Two mutations were previously associated with cardiac conduction blocks and 9 were new mutations (5 absent from ~14'000 control alleles and 4 statistically more prevalent in this BrS cohort than in control alleles). In addition to Brugada, three patients had a bifascicular block and 2 had a complete right bundle branch block. Functional and biochemical studies of 4 selected mutants revealed that these mutations resulted in either a decreased expression (p.Pro779Arg and p.Lys914X) or an increased expression (p.Thr873Ile and p.Leu1075Pro) of TRPM4 channel. TRPM4 mutations account for about 6% of BrS. Consequences of these mutations are diverse on channel electrophysiological and cellular expression. Because of its effect on the resting membrane potential, reduction or increase of TRPM4 channel function may both reduce the availability of sodium channel and thus lead to BrS.

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

Searching the Rhone delta channel in Lake Geneva since François‐Alphonse Forel

In the late 19th century, F.A. FOREL led investigations of the Rhone River delta area of Lake Geneva that resulted in the dis- covery of a textbook example of a river-fed delta system containing impressive subaquatic channels. Well ahead of the marine counterparts, scientific observations and interpretations of water currents shaping the delta edifice for the first time documented how underflow currents carry cold, suspension-laden waters from the river mouth all the way to the deep basin. These early investigations of the Rhone delta laid the basis for follow-up studies in the 20th and 21th centuries. Sediment coring, water-column measurements, manned submersible diving, seismic reflection profiling and bathymetric sur- veying eventually provided a rich database to unravel the key erosional and depositional processes, further documenting the impact of human-induced changes in the catchment. With the merging of old and new scientific knowledge, today a comprehensive understanding prevails of how a delta changes through time, how its channels are formed, and what potential natural hazards may be related to its evolution. New and efficient bathymetric techniques, paired with novel coring operations, provided a time-series of morphologic evolution showing and quantifying the high dynamics of the delta/channel evolution in an unprecedented temporal and spatial reso- lution. Future investigations will continue to further quantify these dynamic processes and to link the evolution of the subaquatic domain with changes and processes in the catchment and with natural hazards. Its size, easy access, and large variety of states and processes will continue to make the Rhone delta area a perfect ‘laboratory’ in which general processes can be studied that could be upscaled or downscaled to other marine and lacustrine deltas.

Sediment dynamics in the subaquatic channel of the Rhone delta (Lake Geneva, France/Switzerland)

bstract With its smaller size, well-known boundary conditions, and the availability of detailed bathymetric data, Lake Geneva’s subaquatic canyon in the Rhone Delta is an excellent analogue to understand sedimentary pro- cesses in deep-water submarine channels. A multidisciplinary research effort was undertaken to unravel the sediment dynamics in the active canyon. This approach included innovative coring using the Russian MIR sub- mersibles, in situ geotechnical tests, and geophysical, sedimentological, geochemical and radiometric analysis techniques. The canyon floor/levee complex is character- ized by a classic turbiditic system with frequent spillover events. Sedimentary evolution in the active canyon is controlled by a complex interplay between erosion and sedimentation processes. In situ profiling of sediment strength in the upper layer was tested using a dynamic penetrometer and suggests that erosion is the governing mechanism in the proximal canyon floor while sedimen- tation dominates in the levee structure. Sedimentation rates progressively decrease down-channel along the levee structure, with accumulation exceeding 2.6 cm/year in the proximal levee. A decrease in the frequency of turbidites upwards along the canyon wall suggests a progressive confinement of the flow through time. The multi-proxy methodology has also enabled a qualitative slope-stability assessment in the levee structure. The rapid sediment loading, slope undercutting and over-steepening, and increased pore pressure due to high methane concentrations hint at a potential instability of the proximal levees. Fur- thermore, discrete sandy intervals show very high methane concentrations and low shear strength and thus could cor- respond to potentially weak layers prone to scarp failures.

Impact of dams, dam removal and dam-related river engineering structures on sediment connectivity and channel morphology of the Fugnitz and the Kaja Rivers

In terms of changing flow and sediment regimes of rivers, dams are often regarded as the most dominant form of human impact on fluvial systems. Dams can decrease the flux of water and sediments leading to channel changes such as upstream aggradation and downstream degradation. The opposite effects occur when dams are removed. Channel degradation often requires further intervention in terms of river bed and bank protection works. The situation evolves more complex in river systems that are impacted by a series of dams due to feedback processes between the different system compartments. A number of studies have recently investigated geomorphic systems using connectivity approaches to improve the understanding of geomorphic system response to change. This paper presents a case study investigating the impact of dam construction, dam removal and dam-related river bed and bank protection measures on the sediment connectivity and channel morphology of the Fugnitz and the Kaja Rivers using a combination of DEM analyses, field surveys and landscape evolution modelling. For both river systems the results revealed low sediment connectivity accompanied by a fine river bed sediment facies in river sections upstream of active dams and of removed dams with protection measures. Contrarily, high sediment connectivity which was accompanied by a coarse river bed sediment facies was observed in river sections either located downstream of active dams or of removed dams with upstream protection. In terms of channel changes, significant channel degradation was examined at locations downstream of active dams and of removed dams. Channel bed and bank protection measures prevent erosion and channel slope recovery after dam removal. Landscape evolution modeling revealed a complex geomorphic response to dam construction and dam removal as sediment output rates and therefore geomorphic processes have been shown to act in a non-linear manner. These insights are deemed to have major implications for river management and conservation, as quality and state of riverine habitats are determined by channel morphology and river bed sediment composition.

Comparison of stab wound probing versus radiological stab wound channel depiction with contrast medium

BACKGROUND Instillation of contrast medium into stab wounds has shown promising results regarding visibility and assessment of general stab direction with computed tomography. However, the accuracy of this method--and, incidentally also probing of stab wounds--has to our knowledge not previously been examined. Also the effect of bluntness of different stabbing objects on the examination of stab wounds was not considered before this study. METHODS Using a pocket-knife, a steak-knife, and a Phillips screwdriver, nine stab wounds each were inflicted to three pork haunches. The depths of the stab wounds were determined by probing and multislice computed tomography (MSCT) after instillation of a contrast medium (CM) and then compared to those observed by dissection, our internal "gold standard". RESULTS In stab wounds inflicted by knives, MSCT-CM and probing provided results which differed by roughly 10-11% from the dissection results. In screwdriver stabs MSCT-CM showed a deviation of almost 30%, probing over 33%. DISCUSSION MSCT-CM is a possible alternative to layer-by-layer dissection in autopsy cases of knife stab wounds. Probing, although obsolete in post-mortem examinations, is sufficiently accurate in determining the length of a stab wound of a living person. In cases of stab wounds with blunt objects such as screwdrivers, neither MSCT-CM nor probing proved to be sufficiently accurate. CONCLUSION MSCT-CM is a possible alternative to layer-by-layer dissection in autopsy cases of knife stab wounds. Probing, although obsolete in post-mortem examinations, is sufficiently accurate in determining the length of a stab wound of a living person. In cases of stab wounds with blunt objects such as screwdrivers, neither MSCT-CM nor probing proved to be sufficiently accurate.

Multi-channel search for squarks and gluinos in sqrts=7 TeV pp collisions with the ATLAS detector

A search for supersymmetric particles in final states with zero, one, and two leptons, with and without jets identified as originating from b-quarks, in 4.7 fb(-1) of root s = 7 TeV pp collisions produced by the Large Hadron Collider and recorded by the ATLAS detector is presented. The search uses a set of variables carrying information on the event kinematics transverse and parallel to the beam line that are sensitive to several topologies expected in supersymmetry. Mutually exclusive final states are defined, allowing a combination of all channels to increase the search sensitivity. No deviation from the Standard Model expectation is observed. Upper limits at 95 % confidence level on visible cross-sections for the production of new particles are extracted. Results are interpreted in the context of the constrained minimal supersymmetric extension to the Standard Model and in supersymmetry-inspired models with diverse, high-multiplicity final states.