Evidence for linkage of adolescent-onset idiopathic generalized epilepsies to chromosome 8-and genetic heterogeneity

Several loci and candidate genes for epilepsies or epileptic syndromes map or have been suggested to map to chromosome 8. We investigated families with adolescent-onset idiopathic generalized epilepsy (IGE), for linkage to markers spanning chromosome 8. The IGEs that we studied included juvenile myoclonic epilepsy (JME), epilepsy with only generalized tonic-clonic seizures occurring either randomly during the day (random grand mal) or on awakening (awakening grand mal), and juvenile absence epilepsy (JAE). We looked for a gene common to all these IGEs, but we also investigated linkage to specific subforms of IGE. We found evidence for linkage to chromosome 8 in adolescent-onset IGE families in which JME was not present. The maximum multipoint LOD score was 3.24 when family members with IGE or generalized spike-and-waves (SW) were considered affected. The LOD score remained very similar (3.18) when clinically normal family members with SW were not considered to be affected. Families with either pure grand mal epilepsy or absence epilepsy contributed equally to the positive LOD score. The area where the LOD score reaches the maximum encompasses the location of the gene for the beta3-subunit of the nicotinic acetylcholine receptor (CHRNB3), thus making this gene a possible candidate for these specific forms of adolescent-onset IGE. The data excluded linkage of JME to this region. These results indicate genetic heterogeneity within IGE and provide no evidence, on chromosome 8, for a gene common to all IGEs.     

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1–NEDD4L WW3 interaction accounts for most of the AMOT–NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW–PPxY core interaction account for the unusually high affinity of the AMOT PPxY1–NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.     

Lack of an association between candidate gene loci and idiopathic generalized epilepsy in Kuwaiti Arab children

Summary Idiopathic generalized epilepsies (IGEs) are the most common types of epilepsy in childhood and adolescence. A variety of data suggest that IGEs have a predominant genetic etiology. Recently, a number of gene mutations have been found to be associated with various types of epilepsy in mainly the Caucasian populations. The objective of this study was to investigate the association of three different candidate genes with IGE in Kuwaiti Arab children. This study includes 123 Kuwaiti patients with a confirmed diagnosis of epilepsy. Most of the patients have had a diagnostic EEG with generalized spike-wave discharges (GSWs). All patients were evaluated by using a validated seizure questionnaire. The clinical type of epilepsy was determined by a trained neurologist/pediatrician. The study also include 100 controls, the control subjects were children which did not have any history of neurological disorders. Blood samples were collected from all patients and control subjects after taking informed consent. DNA was isolated and analyzed by molecular methods. A FokI polymorphism in neuronal nicotinic acetylcholine receptor alpha-4 subunit (CHRNA4) gene was detected by PCR-RFLP method. A missense mutation (Ser248Phe) in CHRNA4 gene was analyzed by PCR-RFLP using HpaII. A C121W mutation in sodium-channel beta-1 subunit (SCN1B) gene was screened by a PCR-RFLP method using HinPI. A 2-bp deletion in Cystatin B gene was detected by PCR-RFLP using XcmI. The incidence of three FokI polymorphism genotypes in Kuwaiti IGE patients was 1,1 (85%), 1,2 (14%) and 2,2 (1%) respectively. The missense mutation Ser248Phe of CHRNA4 gene was not detected at all in Kuwaiti IGE patients. The C387G transversion resulting in C121W change in third exon of the SCN1B gene was detected in 3/123 patients (2%). The patients carrying this mutation also exhibited febrile seizures. The incidence of 2 bp deletion in the cystatin B gene was found to be 4% (5/123 IGE patients). The data obtained from molecular analysis show a lack of association between three candidate genes and clinical expression of IGE in Kuwaiti Arab children. This is completely different from the findings reported from Caucasian populations of France, Australia and USA in which case a strong association has been reported between IGE and these genes. To whom corresspondence should be addressed. Tel: +965-5319486; Fax: +965-5338940; E-mail: haider@hsc.edu.kw     

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

This study describes a method to rapidly screen hundreds of ion channel variants containing non-canonical amino acids. A proof-of-principle introducing photocrosslinking non-canonical amino acids into the human ion channel hASIC1a shows how this approach can provide insights into function and pharmacology.     

Ion channels and epilepsy in man and mouse

Inherited disorders of voltage-gated ion channels are a recently recognized etiology of epilepsy in the developing and mature central nervous system. Two human epilepsy syndromes, benign familial neonatal convulsions and generalized epilepsy with febrile seizures plus, represent K+ and Na+ channelopathies, and other newly defined syndromes have now been mapped to chromosomal regions that are rich in ion channel genes. Experimental mouse models promise a resolution of their intriguing pathophysiology, which includes a diverse array of cellular phenotypes consistent with the differential contributions of individual channels to excitability in neural networks.     

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3- Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.     

Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells

Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.     

The Molecular Biology of Genetic-Based Epilepsies

Epilepsy is one of the most common neurological disorders characterized by abnormal electrical activity in the central nervous system. The clinical features of this disorder are recurrent seizures, difference in age onset, type, and frequency, leading to motor, sensory, cognitive, psychic, or autonomic disturbances. Since the discovery of the first monogenic gene mutation in 1995, it is proposed that genetic factor plays an important role in the mechanism of epilepsy. Genes discovered in idiopathic epilepsies encode for ion channel or neurotransmitter receptor proteins, whereas syndromes with epilepsy as a main feature are caused by genes that are involved in functions such as cortical development, mitochondrial function, and cell metabolism. The identification of these monogenic epilepsy-causing genes provides new insight into the pathogenesis of epilepsies. Although most of the identified gene mutations present a monogenic inheritance, most of idiopathic epilepsies are complex genetic diseases exhibiting a polygenic or oligogenic inheritance. This article reviews recent genetic and molecular progresses in exploring the pathogenesis of epilepsy, with special emphasis on monogenic epilepsy-causing genes, including voltage-gated channels (Na⁺, K⁺, Ca²⁺, Cl^?, and HCN), ligand-gated channels (nicotinic acetylcholine and GABA_A receptors), non-ion channel genes as well as the mitochondrial DNA genes. These progresses have improved our understanding of the complex neurological disorder.     

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor trafficking regulator "stargazin" is related to the claudin family of proteins by Its ability to mediate cell-cell adhesion

Mutations in the Cacng2 gene encoding the neuronal transmembrane protein stargazin result in recessively inherited epilepsy and ataxia in "stargazer" mice. Functional studies suggest a dual role for stargazin, both as a modulatory gamma subunit for voltage-dependent calcium channels and as a regulator of post-synaptic membrane targeting for alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors. Co-immunoprecipitation experiments demonstrate that stargazin can bind proteins of either complex in vivo, but it remains unclear whether it can associate with both complexes simultaneously. Cacng2 is one of eight closely related genes (Cacng1-8) encoding proteins with four transmembrane segments, cytoplasmic termini, and molecular masses between 25 and 44 kDa. This group of Cacng genes constitutes only one branch of a larger monophyletic assembly dominated by over 20 genes encoding proteins known as claudins. Claudins regulate cell adhesion and paracellular permeability as fundamental components of non-neuronal tight junctions. Because stargazin is structurally similar to claudins, we hypothesized that it might also have retained claudin-like functions inherited from a common ancestor. Here, we report that expression of stargazin in mouse L-fibroblasts results in cell aggregation comparable with that produced by claudins, and present evidence that the interaction is heterotypic and calcium dependent. The data suggest that the cell adhesion function of stargazin preceded its current role in neurons as a regulator of either voltage-dependent calcium channels or AMPA receptors. We speculate these complexes may have co-opted the established presence of stargazin at sites of close cell-cell contact to facilitate their own evolving intercellular signaling functions.     

The role of NMDA receptor and neuroligin rare variants in synaptic dysfunction underlying neurodevelopmental disorders

Many genes encoding synaptic proteins are associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorders (ASDs), intellectual disability (ID), and epilepsy. Here we review recent studies on the synaptic effects of disease-associated rare variants identified in two families of synaptic proteins: NMDA receptors (NMDARs) and the postsynaptic adhesion molecules neuroligins (NLGNs). Many NMDAR subunit genes (GRINs) are highly intolerant to variation, and both gain-of-function (GOF) and loss-of-function (LOF) variants are implicated in disease. NLGN genes are also associated with ASDs, and in some cases, contribute to the male bias identified in these patients. Understanding the molecular basis of synaptic dysfunction of rare variants in these genes will help the design of new therapeutic approaches.     

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

Crucial role of the C-terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation

PTEN (phosphatase and tensin homologue deleted on chromosome 10), a potent tumour suppressor and multifunctional signalling protein, is under intricate regulation. In the present study, we have investigated the mechanism and regulation of PTEN ubiquitination catalysed by NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1), a ubiquitin ligase for PTEN we identified recently. Using the reconstituted assay and cellular analysis, we demonstrated that NEDD4-1-mediated PTEN ubiquitination depends on its intact HECT (homologous to E6-associated protein C-terminus) domain. Instead of using its WW domains (protein-protein interaction domains containing two conserved tryptophan residues) as a protein interaction module, NEDD4-1 interacts with PTEN through its N-terminal region containing a C2 domain as well as the HECT domain. Strikingly, we found that a C-terminal truncated PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN, suggesting an intrinsic inhibitory effect of the PTEN C-terminus on PTEN-NEDD4-1 interaction. Moreover, the C-terminal truncated PTEN is more sensitive to NEDD4-1-mediated ubiquitination and degradation. Therefore the present study reveals that the C-terminus of PTEN plays a critical role in stabilizing PTEN via antagonizing NEDD4-1-induced PTEN protein decay; conversely, truncation of the PTEN C-terminus results in rapid NEDD4-1-mediated PTEN degradation, a possible mechanism accounting for attenuation of PTEN function by certain PTEN mutations in human cancers.     

The ENTH domain

The epsin NH2-terminal homology (ENTH) domain is a membrane interacting module composed by a superhelix of alpha-helices. It is present at the NH2-terminus of proteins that often contain consensus sequences for binding to clathrin coat components and their accessory factors, and therefore function as endocytic adaptors. ENTH domain containing proteins have additional roles in signaling and actin regulation and may have yet other actions in the nucleus. The ENTH domain is structurally similar to the VHS domain. These domains define two families of adaptor proteins which function in membrane traffic and whose interaction with membranes is regulated, in part, by phosphoinositides.     

Social effects for locomotion vary between environments in Drosophila melanogaster females

Despite strong purifying or directional selection, variation is ubiquitous in populations. One mechanism for the maintenance of variation is indirect genetic effects (IGEs), as the fitness of a given genotype will depend somewhat on the genes of its social partners. IGEs describe the effect of genes in social partners on the expression of the phenotype of a focal individual. Here, we ask what effect IGEs, and variation in IGEs between abiotic environments, has on locomotion in Drosophila. This trait is known to be subject to intralocus sexually antagonistic selection. We estimate the coefficient of interaction, Ψ, using six inbred lines of Drosophila. We found that Ψ varied between abiotic environments, and that it may vary across among male genotypes in an abiotic environment specific manner. We also found evidence that social effects of males alter the value of a sexually dimorphic trait in females, highlighting an interesting avenue for future research into sexual antagonism. We conclude that IGEs are an important component of social and sexual interactions and that they vary between individuals and abiotic environments in complex ways, with the potential to promote the maintenance of phenotypic variation.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.