Journal: Nature communications

Article Title: Identification of the functional binding pocket for compounds targeting small-conductance Ca 2+ -activated potassium channels

doi: 10.1038/ncomms2017

Figure Lengend Snippet: ( a ) A space-filled model of the CaM-CaMBD2-b complex (3SJQ), depicting the location of PHU (yellow) between the interface of CaM (salmon) and CaMBD2-b (marine) at the CaM N-lobe. Only half of the 2×2 complex is shown for clarity. ( b ) Difference Fourier electron density map constructed using mFo–DFc coefficients calculated in PHENIX prior to modeling PHU and side chain rearrangements that occur in the binding site. The map is contoured at 2.5σ and displayed within a 6 Å sphere of PHU. The map is overlaid with the final coordinates for PHU and its surrounding amino acid residues in the CaM-CaMBD2-a complex. Marine are amino acid residues from CaMBD2-a and salmon are amino acid residues from CaM. Coordinates of the same amino acid residues (gray) from the original CaM-CaMBD2-a complex (without PHU, 1G4Y) are provided for comparison. ( c ) A space-filled model of PHU at the CaM-CaMBD interface. Marine represents CaMBD and salmon represents CaM. ( d ) Comparison of the chemical structure of PHU with that of 1-EBIO and NS309. Both 1-EBIO and NS309 are known SK/IK channel modulators.

Article Snippet: All SK channel modulators were from Tocris, except CyPPA which was from Sigma-Aldrich.

Techniques: Construct, Binding Assay, Comparison

Journal: Nature communications

Article Title: Identification of the functional binding pocket for compounds targeting small-conductance Ca 2+ -activated potassium channels

doi: 10.1038/ncomms2017

Figure Lengend Snippet: ( a ) Raw current traces from an inside-out patch with SK2-a channels expressed. The SK2 channels are activated by 200 nM Ca 2+ , with subsequent potentiation by PHU at the concentrations indicated (all in the presence of 200 nM Ca 2+ ). A voltage ramp, from −100 mV to +100 mV, was applied. ( b ) Dose-response curves for PHU (n = 4) and 1-EBIO (n = 5) for their potentiation of the SK2 channel activities (all in the presence of 200 nM Ca 2+ ). The current amplitudes were measured at −90 mV and normalized to the maximal current obtained either with PHU or 1-EBIO. ( c ) EC50s of potentiation of the SK2 channel activities by PHU (n = 4), 1-EBIO (n = 5), DCEBIO (n = 8), CyPPA (n = 6) and NS309 (n = 3). All modulators were in a 200 nM Ca 2+ solution. Note that the y-axis is in the log scale. ( d ) SK2-a (n = 3) and SK2-b (n = 6) have the same responses to application of NS309. Because SK2-b is less sensitive to Ca 2+ for its activation, 500 nM Ca 2+ was used instead of 200 nM Ca 2+ for SK2-a channels. All data are mean ± s.e.m.

Article Snippet: All SK channel modulators were from Tocris, except CyPPA which was from Sigma-Aldrich.

Techniques: Activation Assay

Journal: Nature communications

Article Title: Identification of the functional binding pocket for compounds targeting small-conductance Ca 2+ -activated potassium channels

doi: 10.1038/ncomms2017

Figure Lengend Snippet: ( a ) Sequence alignment of the fragments (equivalent of E469 to Q487 of SK2-a) of CaMBD at the CaM N-lobe from SK1, SK2, SK3 and IK channels. A477, L480 and V481 are implicated in formation of the binding pocket for the channel modulators from the structure data. Used in the alignment are the human SK1, SK3 and IK channel sequences and the rat SK2 channel sequence. ( b ) A space-filled model of the A477V/L480M mutation (green) with 1-EBIO in the binding pocket. Changes are made based on the structure data using Pymol. Note that T479 points away from 1-EBIO. ( c ) Dose-response curves for potentiation by 1-EBIO of the SK2 channel activities from WT (n = 5), T479S (n = 4) and A477V/L480M (n = 4). 200 nM Ca 2+ was present in all 1-EBIO solutions. ( d ) A477V/L480M mutation significantly increases the potency of 1-EBIO (10-fold). All data are mean ± s.e.m.

Article Snippet: All SK channel modulators were from Tocris, except CyPPA which was from Sigma-Aldrich.

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: Genes, Brain and Behavior

Article Title: A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy

doi: 10.1111/j.1601-183x.2009.00502.x

Figure Lengend Snippet: Figure 3: Schematic diagram of rat Wwox cDNA and the 13-bp deletion of the lde mutant allele. (a) Partial nucleotide sequences of wild type and mutant Wwox cDNA. The deleted sequences of the lde mutant allele are shown in bold type in a rectangle. The lde mutant allele has a 13-bp deletion in exon 9 of the Wwox gene, resulting in a frame shift and abolishing the original termination codon. The deduced amino acid residues are shown below each codon. Aberrant amino acid sequences of the LDE Wwox mutant protein are shown in bold type. Asterisks show the termination codon. (b) PCR amplification of wild type and lde mutant alleles in genomic DNA. Genotypes are shown below the lanes.

Article Snippet: After electrophoresis, separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Pall Fluoro Trans W Membrane, Nippon Genetics, Tokyo, Japan) using a BIO CRAFT BE-300 semi-dry blotting apparatus (Biocraft, Tokyo, Japan) at 14 V for 75 min. After blocking the membrane with Odyssey Blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) overnight at 4◦C, the membranes were incubated with goat anti-Wwox polyclonal antibody (1:100; sc-20528, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-βactin monoclonal antibody (1:5000, Applied Biological Materials Inc., Richmond, BC, Canada), followed by incubation with Alexa Fluor 680 conjugated rabbit anti-goat immunoglobulin G (IgG) antibody (1:20 000; Invitrogen, Carlsbad, CA, USA) and IRDye 800 conjugated donkey anti-mouse IgG antibody (1:20 000; Rockland Inc., Gilbertsville, PA, USA).

Techniques: Mutagenesis

Journal: Genes, Brain and Behavior

Article Title: A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy

doi: 10.1111/j.1601-183x.2009.00502.x

Figure Lengend Snippet: Figure 4: Semi-quantitative RT-PCR analysis of Wwox mRNA expression in the testes and hippocampi of P15 +/+, lde/+ and lde/lde rats. The amount of Wwox cDNA was normalized relative to the amount of Gapdh cDNA in the same sample. Similar levels of expression of Wwox mRNA were observed among all genotypes.

Article Snippet: After electrophoresis, separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Pall Fluoro Trans W Membrane, Nippon Genetics, Tokyo, Japan) using a BIO CRAFT BE-300 semi-dry blotting apparatus (Biocraft, Tokyo, Japan) at 14 V for 75 min. After blocking the membrane with Odyssey Blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) overnight at 4◦C, the membranes were incubated with goat anti-Wwox polyclonal antibody (1:100; sc-20528, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-βactin monoclonal antibody (1:5000, Applied Biological Materials Inc., Richmond, BC, Canada), followed by incubation with Alexa Fluor 680 conjugated rabbit anti-goat immunoglobulin G (IgG) antibody (1:20 000; Invitrogen, Carlsbad, CA, USA) and IRDye 800 conjugated donkey anti-mouse IgG antibody (1:20 000; Rockland Inc., Gilbertsville, PA, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Genes, Brain and Behavior

Article Title: A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy

doi: 10.1111/j.1601-183x.2009.00502.x

Figure Lengend Snippet: Figure 5: Western blotting analysis of Wwox protein in testes and hippocampi of P15 +/+, lde/+ and lde/lde rats. Twenty micrograms of protein extracts was electrophoresed in each lane. Bands of approximately 47 and 42 kDa signal were detected in wild-type and lde/+ protein extracts using anti-Wwox antibody. No signal was detected in the testes and hippocampi of lde/lde protein extracts. The amount of Wwox protein was approximately 20–40% lower in lde/+ than in wild-type testes and hippocampi. Genotypes are shown below the lanes. The faint signals observed around 42 kDa were fluorescence emission leaks from the β-actin signals.

Article Snippet: After electrophoresis, separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Pall Fluoro Trans W Membrane, Nippon Genetics, Tokyo, Japan) using a BIO CRAFT BE-300 semi-dry blotting apparatus (Biocraft, Tokyo, Japan) at 14 V for 75 min. After blocking the membrane with Odyssey Blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) overnight at 4◦C, the membranes were incubated with goat anti-Wwox polyclonal antibody (1:100; sc-20528, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-βactin monoclonal antibody (1:5000, Applied Biological Materials Inc., Richmond, BC, Canada), followed by incubation with Alexa Fluor 680 conjugated rabbit anti-goat immunoglobulin G (IgG) antibody (1:20 000; Invitrogen, Carlsbad, CA, USA) and IRDye 800 conjugated donkey anti-mouse IgG antibody (1:20 000; Rockland Inc., Gilbertsville, PA, USA).

Techniques: Western Blot