NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome.

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.

Neurodevelopmental Disorders Caused by De Novo Variants in KCNB1 Genotypes and Phenotypes.

Importance: Knowing the range of symptoms seen in patients with a missense or loss-of-function variant in KCNB1 and how these symptoms correlate with the type of variant will help clinicians with diagnosis and prognosis when treating new patients. Objectives: To investigate the clinical spectrum associated with KCNB1 variants and the genotype-phenotype correlations. Design, Setting, and Participants: This study summarized the clinical and genetic information of patients with a presumed pathogenic variant in KCNB1. Patients were identified in research projects or during clinical testing. Information on patients from previously published articles was collected and authors contacted if feasible. All patients were seen at a clinic at one of the participating institutes because of presumed genetic disorder. They were tested in a clinical setting or included in a research project. Main Outcomes and Measures: The genetic variant and its inheritance and information on the patient's symptoms and characteristics in a predefined format. All variants were identified with massive parallel sequencing and confirmed with Sanger sequencing in the patient. Absence of the variant in the parents could be confirmed with Sanger sequencing in all families except one. Results: Of 26 patients (10 female, 15 male, 1 unknown; mean age at inclusion, 9.8 years; age range, 2-32 years) with developmental delay, 20 (77%) carried a missense variant in the ion channel domain of KCNB1, with a concentration of variants in region S5 to S6. Three variants that led to premature stops were located in the C-terminal and 3 in the ion channel domain. Twenty-one of 25 patients (84%) had seizures, with 9 patients (36%) starting with epileptic spasms between 3 and 18 months of age. All patients had developmental delay, with 17 (65%) experiencing severe developmental delay; 14 (82%) with severe delay had behavioral problems. The developmental delay was milder in 4 of 6 patients with stop variants and in a patient with a variant in the S2 transmembrane element rather than the S4 to S6 region. Conclusions and Relevance: De novo KCNB1 missense variants in the ion channel domain and loss-of-function variants in this domain and the C-terminal likely cause neurodevelopmental disorders with or without seizures. Patients with presumed pathogenic variants in KCNB1 have a variable phenotype. However, the type and position of the variants in the protein are (imperfectly) correlated with the severity of the disorder.

Structure-Affinity Relationships (SARs) and Structure-Kinetics Relationships (SKRs) of Kv11.1 Blockers.

Kv11.1 (hERG) blockers with comparable potencies but different binding kinetics might display divergent pro-arrhythmic risks. In the present study, we explored structure-kinetics relationships in four series of Kv11.1 blockers next to their structure-affinity relationships. We learned that despite dramatic differences in affinities and association rates, there were hardly any variations in the dissociation rate constants of these molecules with residence times (RTs) of a few minutes only. Hence, we synthesized 16 novel molecules, in particular in the pyridinium class of compounds, to further address this peculiar phenomenon. We found molecules with very short RTs (e.g., 0.34 min for 37) and much longer RTs (e.g., 105 min for 38). This enabled us to construct a k on-k off-KD kinetic map for all compounds and subsequently divide the map into four provisional quadrants, providing a possible framework for a further and more precise categorization of Kv11.1 blockers. Additionally, two representative compounds (21 and 38) were tested in patch clamp assays, and their RTs were linked to their functional IC50 values. Our findings strongly suggest the importance of the simultaneous study of ligand affinities and kinetic parameters, which may help to explain and predict Kv11.1-mediated cardiotoxicity.

Febrile temperatures unmask biophysical defects in Nav1.1 epilepsy mutations supportive of seizure initiation.

Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Nav1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Nav1.1 mutants-R859H (GEFS+) and R865G (DS)-at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Nav1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures.

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue.

AIMS: In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK1) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK1 inhibitors based on the P structure. METHODS AND RESULTS: We analysed seven pentamidine analogues (PA-1 to PA-7) for IK1 blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC50 values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK1 in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 µM. However, PA-6 at 10 µM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 µM. CONCLUSION: PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK1 block.

Dominant missense mutations in ABCC9 cause Cantú syndrome.

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

Biology of cardiac sodium channel Nav1.5 expression.

Na(v)1.5, the pore forming α-subunit of the voltage-dependent cardiac Na(+) channel, is an integral membrane protein involved in the initiation and conduction of action potentials. Mutations in the gene-encoding Na(v)1.5, SCN5A, have been associated with a variety of arrhythmic disorders, including long QT, Brugada, and sick sinus syndromes as well as progressive cardiac conduction defect and atrial standstill. Moreover, alterations in the Na(v)1.5 expression level and/or sodium current density have been frequently noticed in acquired cardiac disorders, such as heart failure. The molecular mechanisms underlying these alterations are poorly understood, but are considered essential for conception of arrhythmogenesis and the development of therapeutic strategies for prevention or treatment of arrhythmias. The unravelling of such mechanisms requires critical molecular insight into the biology of Na(v)1.5 expression and function. Therefore, the aim of this review is to provide an up-to-date account of molecular determinants of normal Na(v)1.5 expression and function. The parts of the Na(v)1.5 life cycle that are discussed include (i) regulatory aspects of the SCN5A gene and transcript structure, (ii) the nature, molecular determinants, and functional consequences of Na(v)1.5 post-translational modifications, and (iii) the role of Na(v)1.5 interacting proteins in cellular trafficking. The reviewed studies have provided valuable information on how the Na(v)1.5 expression level, localization, and biophysical properties are regulated, but also revealed that our understanding of the underlying mechanisms is still limited.

Nav 1.1 dysfunction in genetic epilepsy with febrile seizures-plus or Dravet syndrome.

Relatively few SCN1A mutations associated with genetic epilepsy with febrile seizures-plus (GEFS+) and Dravet syndrome (DS) have been functionally characterized. In contrast to GEFS+, many mutations detected in DS patients are predicted to have complete loss of function. However, functional consequences are not immediately apparent for DS missense mutations. Therefore, we performed a biophysical analysis of three SCN1A missense mutations (R865G, R946C and R946H) we detected in six patients with DS. Furthermore, we compared the functionality of the R865G DS mutation with that of a R859H mutation detected in a GEFS+ patient; the two mutations reside in the same voltage sensor domain of Na(v) 1.1. The four mutations were co-expressed with ß1 and ß2 subunits in tsA201 cells, and characterized using the whole-cell patch clamp technique. The two DS mutations, R946C and R946H, were nonfunctional. However, the novel voltage sensor mutants R859H (GEFS+) and R865G (DS) produced sodium current densities similar to those in wild-type channels. Both mutants had negative shifts in the voltage dependence of activation, slower recovery from inactivation, and increased persistent current. Only the GEFS+ mutant exhibited a loss of function in voltage-dependent channel availability. Our results suggest that the R859H mutation causes GEFS+ by a mixture of biophysical defects in Na(v) 1.1 gating. Interestingly, while loss of Na(v) 1.1 function is common in DS, the R865G mutation may cause DS by overall gain-of-function defects.

Inhibition of lysosomal degradation rescues pentamidine-mediated decreases of K(IR)2.1 ion channel expression but not that of K(v)11.1.

The antiprotozoal drug pentamidine inhibits two types of cardiac rectifier potassium currents, which can precipitate life-threatening arrhythmias. Here, we use pentamidine as a tool to investigate whether a single drug affects trafficking of two structurally different potassium channels by identical or different mechanisms, and whether the adverse drug effect can be suppressed in a channel specific fashion. Whole cell patch clamp, Western blot, real time PCR, and confocal laser scanning microscopy were used to determine potassium current density, ion channel protein levels, mRNA expression levels, and subcellular localization, respectively. We demonstrate that pentamidine inhibits delayed (I(Kr)) and inward (I(K1)) rectifier currents in cultured adult canine cardiomyocytes. In HEK293 cells, pentamidine inhibits functional K(v)11.1 channels, responsible for I(Kr), by interfering at the level of full glycosylation, yielding less mature form of K(v)11.1 at the plasma membrane. In contrast, total K(IR)2.1 expression levels, underlying I(K1), are strongly decreased, which cannot be explained from mRNA expression levels. No changes in molecular size of K(IR)2.1 protein were observed, excluding interference in overt glycosylation. Remaining K(IR)2.1 protein is mainly expressed at the plasma membrane. Inhibition of lysosomal protein degradation is able to partially rescue K(IR)2.1 levels, but not those of K(v)11.1. We conclude that 1) a single drug can interfere in cardiac potassium channel trafficking in a subtype specific mode and 2) adverse drug effects can be corrected in a channel specific manner.

Exploring chemical substructures essential for HERG k(+) channel blockade by synthesis and biological evaluation of dofetilide analogues.

In this study we followed a new approach to analyze molecular substructures required for hERG channel blockade. We designed and synthesized 40 analogues of dofetilide (1), a potent hERG potassium channel blocker, and established structure-activity relationships (SAR) for their interaction with this important cardiotoxicity-related off-target. Structural modifications to dofetilide were made by diversifying the substituents on the phenyl rings and the protonated nitrogen and by varying the carbon chain length. The analogues were evaluated in a radioligand binding assay and SAR data were derived with the aim to specify structural features that give rise to hERG toxicity.

Functional analysis of novel KCNQ2 mutations found in patients with Benign Familial Neonatal Convulsions.

Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant alpha-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.

Fever-induced QTc prolongation and ventricular arrhythmias in individuals with type 2 congenital long QT syndrome.

Type 2 congenital long QT syndrome (LQT-2) is linked to mutations in the human ether a-go-go-related gene (HERG) and is characterized by rate-corrected QT interval (QTc) prolongation, ventricular arrhythmias, syncope, and sudden death. Recognized triggers of these cardiac events include emotional and acoustic stimuli. Here we investigated the repeated occurrence of fever-induced polymorphic ventricular tachycardia and ventricular fibrillation in 2 LQT-2 patients with A558P missense mutation in HERG. ECG analysis showed increased QTc with fever in both patients. WT, A558P, and WT+A558P HERG were expressed heterologously in HEK293 cells and were studied using biochemical and electrophysiological techniques. A558P proteins showed a trafficking-deficient phenotype. WT+A558P coexpression caused a dominant-negative effect, selectively accelerated the rate of channel inactivation, and reduced the temperature-dependent increase in the WT current. Thus, the WT+A558P current did not increase to the same extent as the WT current, leading to larger current density differences at higher temperatures. A similar temperature-dependent phenotype was seen for coexpression of the trafficking-deficient LQT-2 F640V mutation. We postulate that the weak increase in the HERG current density in WT-mutant coassembled channels contributes to the development of QTc prolongation and arrhythmias at febrile temperatures and suggest that fever is a potential trigger of life-threatening arrhythmias in LQT-2 patients.

Connexin43 repression following epithelium-to-mesenchyme transition in embryonal carcinoma cells requires Snail1 transcription factor.

Embryonic stem (ES) cells and embryonal carcinoma (EC) cells express high amounts of functional connexin43 (Cx43). During mesoderm formation and subsequent cardiac differentiation, Cx43 is initially down-regulated but is up-regulated again as the emerging cardiomyocytes mature. In this study, we investigated the regulation of Cx43 expression during early phases of differentiation in F9 and P19 EC cells. We found a striking inverse correlation between the expression of Cx43 and that of the transcriptional repressor Snail1. No clear relationship was found with Smad-interacting-protein1 (SIP1), another transcription factor inducing epithelium-to-mesenchyme transition (EMT). Promoter-reporter assays indicated Cx43 repression at the promoter level by ectopically expressed Snail1. To establish whether the Cx43 down-regulation depends on endogenous Snail1, MES-1 cells, differentiated derivatives of P19 EC, were stably transfected by an siRNA construct silencing Snail1 expression. This resulted in a mesenchyme-to-epithelium transition, which was accompanied by increased levels of Cx43 mRNA and protein and enhanced metabolic and electrical coupling. We conclude that Snail1-mediated EMT results in a Cx43 repression.

Cloning, embryonic expression, and functional characterization of two novel connexins from Xenopus laevis.

Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays > 60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.

Pro-arrhythmogenic potential of immature cardiomyocytes is triggered by low coupling and cluster size.

OBJECTIVE: Cell transplantation strategies to regenerate compromised myocardium take advance of in vitro generated cardiomyocytes. Common in those immature myocytes is spontaneous impulse formation and a restricted ability to establish proper electrical interaction. Spontaneous impulse formation and impaired cell-to-cell coupling have been shown to be arrhythmogenic. To investigate whether these features harbour a pro-arrhyhmogenic potential for cell transplantation, a co-culture of spontaneously active neonatal rat cardiomyocytes (NRC) and quiescent adult dog cardiomyocytes (ADC) was used. METHODS: ADCs and NRCs were isolated and cultured on laminin-coated substrates. Connexin43, N-cadherin and alpha-actinin expression was evaluated with immunohistochemistry. Intercellular coupling was measured in cell pairs using the dual voltage clamp technique and fluorescent dye injection. RESULTS: One day after isolation, NRCs were beating spontaneously, while ADCs remained quiescent in monoculture. ADC resting membrane potential was -80.3+/-0.2 mV (mean+/-SEM, N=24) and did not change significantly over time. NRCs had a maximal diastolic potential of -65.0+/-2.8 mV (N=4). After one day of co-culture, pseudopodia-like extensions developed at the former intercalated discs of ADCs, contacting the NRCs. Only ADCs that contacted three or more NRCs started to beat in synchrony. Expression of connexin43 and N-cadherin indicated presence of electrical and mechanical junctions at the interface between the two cell-types. Transfer of Lucifer Yellow demonstrated junctional permeability between ADCs and NRCs. Junctional conductance between ADC-ADC (31.9+/-5.1 nS, N=10) and NRC-NRC (35.0+/-9.6 nS, N=6) pairs was significantly higher compared to ADC-NRC pairs (9.7+/-2.9 nS, N=8). Gap-junctional blockade with halothane reversibly abolished NRC-triggered beating of ADCs. Computer simulations demonstrated that within a delicate 'window' of gap junctional conductance small clusters of spontaneously active cells are able to induce triggered activity in quiescent mature myocytes but also in a two-dimensional sheet of ventricular cells. CONCLUSION: Spontaneously active immature cardiomyocytes are able to trigger mature cardiomyocytes depending on their level of electrical coupling and the amount of coupled immature myocytes.

Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis.

Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals.

A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells.

The clinical application of stem cell therapies is still limited by the ability to produce defined, differentiated cell populations in large numbers in culture. High throughput screens to identify factors which enhance differentiation to particular lineages and promote expansion of precursors in culture are dependent on the development of sensitive and reproducible assays for screening. Here we describe a bioassay to identify factors with cardiomyogenic activity which enhance the yield of cardiomyocytes from undifferentiated stem cells. The assay is based on a Green Fluorescent Protein (GFP) reporter under the transcriptional control of the 250 bp MLC-2v promoter expressed in pluripotent P19 embryonal carcinoma cells. We show that reporter expression is limited to developing cardiomyocytes and can be used to determine quantitatively the number of ventricular cardiomyocytes formed in cultures under inducing or non-inducing conditions. This assay differs from all others described previously in that it has an easily quantifiable readout, there is negligible background differentiation in the absence of exogenous cardiogenic factors and it is carried out feeder cell-free. Thus, it is entirely independent of competing differentiation inhibitory factors, such as leukemia inhibitory factor. Patch clamp electrophysiology of the GFP-positive cells confirmed their functional ventricular phenotype and indicated that selection on the basis of GFP would provide cells suitable for transplantation.

Trafficking and functional expression of cardiac Na+ channels.

Voltage-gated Na(+)channels are essential for the amplitude and upstroke velocity of the cardiac action potential, which are important determinants for impulse propagation and impulse conduction velocity of throughout the working myocardium. Mutations in the major cardiac Na(+)channel gene SCN5A have been implicated in rare, familial forms of cardiac arrhythmias, namely LQT3, Brugada syndrome, progressive cardiac conduction disorder and sudden infant death syndrome. It is increasingly recognized that such mutations--apart from changing channel gating characteristics--may also be related to changes in channel protein trafficking and expression. Regulation of ion channel protein expression depends on a fine-tuned balance among various processes, such as gene transcription, RNA processing, protein synthesis, assembly and post-translational modification, the transport to the cell surface, the anchoring to the cytoskeleton, and regulation of endocytosis and controlled degradation of the protein. During the last decade, interest in factors that control the expression level of ion channels and mechanisms that are involved in targeting of channel proteins to specific sub-cellular and membrane domains is increasing. This review focuses on the current knowledge of mechanisms of cardiac Na(+) channel protein trafficking and expression in cardiomyocytes and its relation to Na(+)-channelopathies.

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes.

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.