The isolation of the peptide inhibitor of M-type K+ current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., ...Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277–280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K+ channels was also studied. BeKm-1 inhibited hERG1 channels with an IC50 of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.
AF276623
Previous studies have explored the use of departure from Hardy-Weinberg equilibrium (DHW) for fine mapping Mendelian disorders and for general fine mapping. Other studies have used Hardy-Weinberg ...tests for genotyping quality control. To enable investigators to make rational decisions about whether DHW is due to genotyping error or to underlying biology, we developed an analytic framework and software to determine the parameter values for which DHW might be expected for common diseases. We show analytically that, for a general disease model, the difference between population and Hardy-Weinberg–expected genotypic frequencies (Δ) at the susceptibility locus is a function of the susceptibility-allele frequency (
q), heterozygote relative risk (β), and homozygote relative risk (γ). For unaffected control samples, Δ is a function of risk in nonsusceptible homozygotes (α), the population prevalence of disease (
K
P
),
q, β, and γ. We used these analytic functions to calculate Δ and the number of cases or controls needed to detect DHW for a range of genetic models consistent with common diseases (1.1 ≤ γ ≤ 10 and 0.005 ≤
K
P
≤ 0.2). Results suggest that significant DHW can be expected in relatively small samples of patients over a range of genetic models. We also propose a goodness-of-fit test to aid investigators in determining whether a DHW observed in the context of a case-control study is consistent with a genetic disease model. We illustrate how the analytic framework and software can be used to help investigators interpret DHW in the context of association studies of common diseases.
The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective
action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 ...shares the common molecular scaffold with other short scorpion
toxins. The toxin spatial structure resolved by NMR consists of a short α-helix and a triple-stranded antiparallel β-sheet.
By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K + (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the α-helix and following loop
whereas the âtraditionalâ functional site of other short scorpion toxins is formed by residues from the β-sheet. Thus the
unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not
Tourette's syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of ...definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association study (GWAS) of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel and French Canadians from Quebec, Canada. In a primary meta-analysis of GWAS data from these European ancestry samples, no markers achieved a genome-wide threshold of significance (P<5 × 10(-8)); the top signal was found in rs7868992 on chromosome 9q32 within COL27A1 (P=1.85 × 10(-6)). A secondary analysis including an additional 211 cases and 285 controls from two closely related Latin American population isolates from the Central Valley of Costa Rica and Antioquia, Colombia also identified rs7868992 as the top signal (P=3.6 × 10(-7) for the combined sample of 1496 cases and 5249 controls following imputation with 1000 Genomes data). This study lays the groundwork for the eventual identification of common TS susceptibility variants in larger cohorts and helps to provide a more complete understanding of the full genetic architecture of this disorder.
A 600 MHz 1H NMR study of toxin OSK1, blocker of small-conductance Ca2+-activated K+ channels, is presented. The unambiguous sequential assignment of all the protons of the toxin was obtained using ...TOCSY, DQF-COSY, and NOESY experiments at pH 3.0 (10, 30, and 45 °C) in aqueous solution. 3 J N α, 3 J αβ vicinal spin coupling constants were determined in high-resolution spectra. The cross-peak volumes in NOESY spectra and the coupling constants were used to define the local structure of the protein by the program HABAS and to generate torsion angle and interproton distance constraints for the program DIANA. Hydrogen−deuterium exchange rates of amide protons showed possible locations of hydrogen bonds. The hydrogen bond acceptors and disulfide bridges between residues 8−28, 14−33, and 18−35 were determined when analyzing distance distribution in preliminary DIANA structures. All constraints were used to obtain a set of 30 structures by DIANA. The resulting rms deviations over 30 structures are 1.30 Å for the heavy atoms and 0.42 Å for the backbone heavy atoms. The structures were refined by constrained energy minimization using the SYBYL program. Their analysis indicated the existence of the α-helix (residues 10−21) slightly distorted at the Cys14 residue, two main strands of the antiparallel β-sheet (24−29, 32−38), and the extended fragment (2−6). The motif is stabilized by the disulfide bridges in the way, common to all known scorpion toxins. Using the fine spatial toxin structure, alignment of the homologues, mutagenesis analysis, and comparison of scorpion toxin family functions, we delineate some differences significant for the toxin specificity.
The results of developing a compact version of the Precision Laser Inclinometer (CPLI) with the reduced overall dimensions of 20 × 20 × 20 cm and weight of 10 kg are presented. Experimental data on ...detected angular oscillations of the Earth’s surface at the JINR site are obtained. The achieved sensitivity is 6 × 10
–11
rad/Hz
1/2
in the frequency range 1.4 × 10
–3
–10 Hz. The CPLI can be used in modern physical experiments for seismic isolation of large-scale installations. Reduction of the impact of microseismic angular oscillations of the Earth’s surface on the sensitive elements of the VIRGO Interference Gravitational Antenna, the Large Hadron Collider, and NICA will increase the accuracy of the experiments.
Highly purified α-latrotoxin from the black widow spider venom (α-LTX) consists of two polypeptides with mol. wts of 130,000 and 8000 (LMWP). We have isolated two low mol. wt proteins LMWP and LMWP2 ...from the low mol. wt fraction of this venom. The chemical properties of these proteins and partial amino acid sequence of novel protein LMWP2 were studied. By means of i.v. or intracerebroventricular injections into mice it was shown that low mol. wt components of the venom at concentrations of 2.3 mg/kg and 0.8 mg/kg, respectively, did not possess any direct toxic effect on vertebrates. Injections of each protein into the third thoracic segment of cockroaches
Periplaneta americana (doses up to 80 μg/g) did not cause lethality or paralysis of insects.
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