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14-3-3θ is a binding partner of rat Eag1 potassium channels.

Hsu PH, Miaw SC, Chuang CC, Chang PY, Fu SJ, Jow GM, Chiu MM, Jeng CJ - PLoS ONE (2012)

Bottom Line: One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain.Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1.Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

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Endogenous expression of 14-3-3θ and rEag1 in neurons.(A) Co-immunoprecipitation of 14-3-3θ and rEag1. Detergent solubilized proteins from the lysates of rat forebrain were immunoprecipitated (IP) with the anti-14-3-3θ (upper panel) or the anti-rEag1 antibody (lower panel), followed by immunoblotting (WB) analyses with the anti-14-3-3θ or the anti-rEag1 antibody. The non-immune mouse or rabbit IgG was used in parallel as negative control. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The arrowhead and arrow refers to the protein bands of 14-3-3θ and rEag1, respectively. (B) Immunofluorescence staining of rEag1 (left panels) and 14-3-3θ (middle panels) in cultured hippocampal neurons. The area highlighted in the white boxes is viewed under a higher magnification (I, II). Arrows label the sites of co-localization of 14-3-3θ and rEag1 (right panels), which displayed significant punctuate patterns over a wide region along the neurites. Scale bar, 25 µm. These co-immunoprecipitation and immunofluorescence data are representative of four to seven independent experiments.
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pone-0041203-g005: Endogenous expression of 14-3-3θ and rEag1 in neurons.(A) Co-immunoprecipitation of 14-3-3θ and rEag1. Detergent solubilized proteins from the lysates of rat forebrain were immunoprecipitated (IP) with the anti-14-3-3θ (upper panel) or the anti-rEag1 antibody (lower panel), followed by immunoblotting (WB) analyses with the anti-14-3-3θ or the anti-rEag1 antibody. The non-immune mouse or rabbit IgG was used in parallel as negative control. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The arrowhead and arrow refers to the protein bands of 14-3-3θ and rEag1, respectively. (B) Immunofluorescence staining of rEag1 (left panels) and 14-3-3θ (middle panels) in cultured hippocampal neurons. The area highlighted in the white boxes is viewed under a higher magnification (I, II). Arrows label the sites of co-localization of 14-3-3θ and rEag1 (right panels), which displayed significant punctuate patterns over a wide region along the neurites. Scale bar, 25 µm. These co-immunoprecipitation and immunofluorescence data are representative of four to seven independent experiments.

Mentions: Given that both 14-3-3θ and rEag1 proteins are abundantly expressed in the brain, it is imperative to determine whether the potential interaction of these two proteins can also be verified in neurons. Crude membrane (P2) fractions prepared from rat forebrain homogenates were subject to immunoprecipitation with the anti-14-3-3θ antibody, followed by immunoblotting with the anti-rEag1 antibody. As demonstrated in Figure 5A, rEag1 was effectively co-immunoprecipitated with 14-3-3θ, suggesting that in the rat brain, endogenous 14-3-3θ and rEag1 co-existed in the same protein complex.


14-3-3θ is a binding partner of rat Eag1 potassium channels.

Hsu PH, Miaw SC, Chuang CC, Chang PY, Fu SJ, Jow GM, Chiu MM, Jeng CJ - PLoS ONE (2012)

Endogenous expression of 14-3-3θ and rEag1 in neurons.(A) Co-immunoprecipitation of 14-3-3θ and rEag1. Detergent solubilized proteins from the lysates of rat forebrain were immunoprecipitated (IP) with the anti-14-3-3θ (upper panel) or the anti-rEag1 antibody (lower panel), followed by immunoblotting (WB) analyses with the anti-14-3-3θ or the anti-rEag1 antibody. The non-immune mouse or rabbit IgG was used in parallel as negative control. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The arrowhead and arrow refers to the protein bands of 14-3-3θ and rEag1, respectively. (B) Immunofluorescence staining of rEag1 (left panels) and 14-3-3θ (middle panels) in cultured hippocampal neurons. The area highlighted in the white boxes is viewed under a higher magnification (I, II). Arrows label the sites of co-localization of 14-3-3θ and rEag1 (right panels), which displayed significant punctuate patterns over a wide region along the neurites. Scale bar, 25 µm. These co-immunoprecipitation and immunofluorescence data are representative of four to seven independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401112&req=5

pone-0041203-g005: Endogenous expression of 14-3-3θ and rEag1 in neurons.(A) Co-immunoprecipitation of 14-3-3θ and rEag1. Detergent solubilized proteins from the lysates of rat forebrain were immunoprecipitated (IP) with the anti-14-3-3θ (upper panel) or the anti-rEag1 antibody (lower panel), followed by immunoblotting (WB) analyses with the anti-14-3-3θ or the anti-rEag1 antibody. The non-immune mouse or rabbit IgG was used in parallel as negative control. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The arrowhead and arrow refers to the protein bands of 14-3-3θ and rEag1, respectively. (B) Immunofluorescence staining of rEag1 (left panels) and 14-3-3θ (middle panels) in cultured hippocampal neurons. The area highlighted in the white boxes is viewed under a higher magnification (I, II). Arrows label the sites of co-localization of 14-3-3θ and rEag1 (right panels), which displayed significant punctuate patterns over a wide region along the neurites. Scale bar, 25 µm. These co-immunoprecipitation and immunofluorescence data are representative of four to seven independent experiments.
Mentions: Given that both 14-3-3θ and rEag1 proteins are abundantly expressed in the brain, it is imperative to determine whether the potential interaction of these two proteins can also be verified in neurons. Crude membrane (P2) fractions prepared from rat forebrain homogenates were subject to immunoprecipitation with the anti-14-3-3θ antibody, followed by immunoblotting with the anti-rEag1 antibody. As demonstrated in Figure 5A, rEag1 was effectively co-immunoprecipitated with 14-3-3θ, suggesting that in the rat brain, endogenous 14-3-3θ and rEag1 co-existed in the same protein complex.

Bottom Line: One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain.Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1.Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

Show MeSH
Related in: MedlinePlus