Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Digitized Phosphorimager scan of SDS-PAGE analysis of [ 35 S] Met/Cys-labeled channels (KCNQ2; KCNQ2/3 and KCNQ3) and syntaxin 1A ( syx ) proteins coprecipitated by the corresponding antibodies from 1% Triton X-100 homogenates of whole oocytes, that were injected with the channels mRNA alone or with syntaxin 1A mRNA alone or coinjected with the channels and syntaxin 1A mRNAs (as indicated above the lanes). The protein samples were analyzed on an 8% gel. Arrows indicate the relevant proteins. B, KCNQ3 and KCNQ1 channels do not interact with syntaxin 1A as strongly as KCNQ2 in oocytes. Left panel : The channels and syntaxin proteins coprecipitated by the corresponding antibodies (as indicated below the lanes). Right panel : Reciprocal coimmunoprecipitation in oocytes from the same experiment, carried out using a monoclonal syntaxin 1A antibody (IP syx). C, Interaction of KCNQ2, KCNQ2\3, KCNQ3 and KCNQ1 with syntaxin 1A. Bars depict ratios (quantified by ImageQuant) of syntaxin to the channels, precipitated by the corresponding channel antibodies. Numbers in parentheses refer to number of oocyte batches. *p<0.05.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: SDS Page, Labeling, Injection

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Syntaxin 1A (syx) reduces current amplitudes of KCNQ2 (Aa) and KCNQ2\3 (Ba) channels, expressed in Xenopus oocytes, but not those of KCNQ3* (Ca). Representative current traces from single oocytes of the same batch injected with the channels mRNA alone or with syntaxin 1A mRNA (0.75 ng/oocyte; +syx). Inset: the voltage protocol used to elicit currents. B, Syntaxin 1A reduces the maximal conductances of KCNQ2 (Ab), and KCNQ2\3 (Bb), but not of KCNQ3* (Cb). Conductance-Voltage (G–V) relationships for the channels in the presence and absence of syntaxin 1A, normalized to the maximal conductance in the absence of syntaxin 1A or each normalized to itself (inset). G values were obtained from peak currents, assuming a reversal potential of −98 mV for K+ ions. Two component Boltzmann equation G/G max = 1/(1+exp(−(V 1/2 −V)/a), ,was fitted to the data. C, Syntaxin 1A slows down only the rate of activation of KCNQ2 (Ac) but not of KCNQ2\3 (Bc) or KCNQ3* (Cc). Inset: overlay of representative traces elicited at +5 mV showing the activation of KCNQ2 currents in the presence and absence of syntaxin 1A. The rising phase of the currents elicited at all denoted potentials was fitted by a bi-exponential function, deriving fast and slow time constants (τ fast and τ slow). Data in B and C were averaged from three oocyte batches with at least 5 oocytes per batch. *p<0.05, **p<0.01.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Injection, Activation Assay

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Schematic representation of the C-terminus of KCNQ2, in which the syntaxin 1A ( syx ) binding domain is indicated. B, Top : Interaction of syntaxin 1A with GST fusion proteins corresponding to different parts of the C-termini of KCNQ2 and KCNQ3. In vitro synthesized 35 S labeled syntaxin 1A was reacted with the indicated GST fusion proteins. Bottom : Coomassie blue (CB) staining of the protein gel. Numbers denote molecular weight markers. The bar diagram shows the normalized syntaxin 1A binding values. The intensity of the immunoreactive band of syntaxin 1A ( syx ) in different groups was normalized to the corresponding intensity of the coomassie blue ( CB ) staining of the peptide. The data were averaged from several independent experiments and includes the binding of syntaxin 1A to helices A, A+B and A+B+C. C, Top : Stochiometry of the binding of syntaxin 1A to helix A of KCNQ2, derived from binding curves that show saturation. Recombinant hexahistidine-tagged (His 6 ) cytoplasmic part of syntaxin 1A at the indicated concentrations was bound to immobilized GST-helix A (150 pmol) in a 1 ml reaction volume. Bound syntaxin 1A was determined by SDS-PAGE and immunoblotting with syntaxin 1A antibody (inset). ECL signal intensities were quantified with TINA software and converted to picomoles by the use of standard curves for the corresponding proteins. Bottom : Calibration gel which demonstrates the amount of syntaxin 1A coprecipitated in the experiment. Unbound recombinant hexahistidine-tagged (His 6 ) cytoplasmic part of syntaxin 1A at the indicated concentrations was loaded on an 8% gel and immunoblotted with syntaxin 1A antibody. D, helix A (aa 339–360) is required for syntaxin 1A's binding to KCNQ2. Oocytes were injected with syntaxin 1A mRNA alone or co-injected with syntaxin 1A and KCNQ2/Δ339–360 deletion mutant/Δ372–493 deletion mutant. The binding assay was performed as described.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Binding Assay, In Vitro, Synthesized, Labeling, Staining, Molecular Weight, Derivative Assay, Recombinant, SDS Page, Western Blot, Software, Injection, Mutagenesis

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Syntaxin 1A ( syx ) coprecipitates with KCNQ2 from 2% Chaps synaptosomal lysate by anti-KCNQ2 antibody. Lysates were incubated with KCNQ2 antibody in the absence or presence (+peptide) of antigen peptide-HC (heavy chain). Numbers indicate molecular weight markers. B, GST-syntaxin 1A fusion protein “pulls down” KCNQ2 from synaptosomal lysates. Syx-GST (corresponding to the cytosolic part of syntaxin 1A) or GST immobilized on GSH-agarose beads (each at 150 pmoles) were incubated with 2% CHAPS lysate (200 µg) for 12 h at 4°C. Precipitated proteins were separated by SDS-gel (8% polyacrylamide) and immunoblotted with anti-KCNQ2 antibody (upper panel). The lower panel shows a Ponceau S staining of the blot, which demonstrates the equal protein loading of syntaxin 1A-GST and GST proteins. C, The binding of syntaxin 1A to helices A+B+C of KCNQ2 is stronger than its binding to the same helices in KCNQ3. Syntaxin 1A coprecipitated with KCNQ2 and KCNQ3 from 2% Chaps synaptosomal lysate by anti-KCNQ2/KCNQ3 antibody. Precipitated proteins were separated by SDS-gel (8% polyacrylamide) and immunoblotted with anti-syntaxin 1A antibody.

Article Snippet: The primary antibodies used were KCNQ2-C terminus, KCNQ3-C terminus (Alomone Labs, Jerusalem, Israel) and monoclonal anti-HPC-1 (Sigma Israel, Rehovot, Israel). cDNAs and mRNAs of the chimeric channels with different transmembrane segments were described , .

Techniques: Incubation, Molecular Weight, SDS-Gel, Staining, Binding Assay