Idiopathic generalized epilepsies account for about 40% of epilepsy up to age 40 and commonly have a genetic basis. One type is benign familial neonatal convulsions (BFNC), a dominantly inherited disorder of newborns. We have identified a sub-microscopic deletion of chromosome 20q13.3 that co-segregates with seizures in a BFNC family. Characterization of cDNAs spanning the deleted region identified one encoding a novel voltage-gated potassium channel, KCNQ2, which belongs to a new KQT-like class of potassium channels. Five other BFNC probands were shown to have KCNQ2 mutations, including two transmembrane missense mutations, two frameshifts and one splice-site mutation. This finding in BFNC provides additional evidence that defects in potassium channels are involved in the mammalian epilepsy phenotype.

Alzheimer's disease (AD) is characterized by the deposition of amyloid plaques in the parenchyma and vasculature of the brain. Although previous analytical studies have provided much information about the composition and structure of synthetic amyloid-beta fibrils, there is, surprisingly, a dearth of data on intact amyloid plaques from AD brain. Therefore, to elucidate the structure and detailed composition of isolated amyloid plaque cores, we utilized a high-resolution, nondestructive technique, Raman microscopy. The data are of very high quality and contain detailed information about protein composition and conformation, about post-translational modification, and about the chemistry of metal binding sites. Remarkably, spectra obtained for senile plaque (SP) cores isolated from AD brain are essentially identical both within and among brains. The Raman data show for the first time that the SP cores are composed largely of amyloid-beta and confirm inferences from X-ray studies that the structure is beta-sheet with the additional possibility that this may be present as a parallel beta-helix. Raman bands characteristic of methionine sulfoxide show that extensive methionine oxidation has occurred in the intact plaques. The Raman spectra also demonstrate that Zn(II) and Cu(II) are coordinated to histidine residues in the SP cores, at the side chains' N(tau) and N(pi) atoms, respectively. Treatment of the senile plaques with the chelator ethylenediaminetetraacetate reverses Cu binding to SP histidines and leads to a broadening of amide features, indicating a "loosening" of the beta-structure. Our results indicate that Abeta in vivo is a metalloprotein, and the loosening of the structure following chelation treatment suggests a possible means for the solubilization of amyloid deposits. The results also reveal a direct chemical basis for oxidative damage caused by amyloid-beta protein in AD.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

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The toxin 3-nitropropionic acid 5 is produced by certain plants and fungi. It is a specific inhibitor of mitochondrial respiratory complex II. Fatalities after eating moldy sugarcane have been linked to 3-NP toxicity (1, 2). Ruminants grazing in regions with 3-NP-producing plants acquire resistance because of reduction of the nitro group to an amine by ruminal bacteria (3).The effectiveness of 3-NP in vivo after injection or oral administration has made it useful in studies involving tissues or whole animals. Ingestion of 3-NP results in neurodegeneration with symptoms resembling those of Huntington disease (4), and conversely Huntington disease results in a loss of complex II activity (5); thus 3-NP has been used to produce an animal model for the study of Huntington disease (6, 7). Symptoms also include convulsions, and 3-NP is being looked at for inducing a model of epilepsy (8). Prior subacute 3-NP poisoning seems to provide resistance to ischemic damage to nervous tissue by a preconditioning effect (9) similar to that resulting from mild ischemia.The target of 3-NP is Complex II, which is both a member of the Krebs tricarboxylic acid cycle (oxidizing succinate to fumarate) and an entry point for electrons into the respiratory chain at the level of ubiquinol. It consists of a large flavoprotein subunit containing covalently bound FAD, an iron-sulfur protein (IP) with three different iron-sulfur clusters, and two small membrane anchor subunits (chains C and D) ligating a single low spin heme of type B. Human genetic defects in the IP subunits or chains C or D lead to development of paragangliomas (10, 11). A mutation in chain C leads to premature aging in nematodes, presumably through excessive production of free radicals (12). Bacterial homologs succinate:quinone oxidoreductase (SQR) and menaquinol: fumarate oxidoreductase (FRD) in Escherichia coli have been studied as genetically manipulable models for the mitochondrial protein. Recent reviews cover this family of enzymes (13-18). X-ray crystallographic structures are available for a number of members of the family. The available mitochondrial structures and representative bacterial examples are listed in Table 1.The toxin 3-NP, structurally similar to and isoelectronic with the substrate succinate, is believed to be a suicide inactivator of Complex II. Alston et al. (19) proposed, based on previous observations and on their own experience with another flavoprotein, that the normal reaction pathway involves a temporary adduct with the N-5 nitrogen of flavin, which in the case of 3-NP collapses to a stable adduct resulting in permanent inactivation. Irreversible covalent modification of the flavin was ruled out by later work (20) examining the spectral change induced and showing that unmodified flavin peptide could be isolated from the inactivated complex by mild proteolysis. It was proposed that 3-NP is oxidized to 3-nitroacrylate, an unstable molecule that then reacts with some residue in the active site. A cysteine that was believed to be in the active ...

Benign familial neonatal convulsions (BFNC) is a rare autosomal dominant generalized epilepsy of the newborn infant. Seizures occur repeatedly in the first days of life and remit by approximately 4 months of age. Previously our laboratory cloned two novel potassium channel genes, KCNQ2 and KCNQ3, and showed that they are mutated in patients with BFNC. In this report, we characterize the breakpoints of a previously reported interstitial deletion in the KCNQ2 gene and show that only KCNQ2 is deleted. We identify 11 novel mutations in KCNQ2 and one novel mutation in the KCNQ3 potassium channel genes. In one family, the phenotype extends beyond neonatal seizures and includes rolandic seizures, and a subset of families has onset of seizures in infancy. In the Xenopus oocyte expression system, we characterize five KCNQ2 and one KCNQ3 disease-causing mutations. These mutations cause a variable loss of function, and selective effects on the biophysical properties of KCNQ2/KCNQ3 heteromultimeric channels. We report here the first dominant negative mutation in KCNQ2 that has a phenotype of neonatal seizures without permanent clinical CNS impairment.

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Although only about one third of patients with a first unprovoked seizure will have further seizures within five years, about three quarters of those with two or three unprovoked seizures have further seizures within four years.