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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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Localization of 14-3-3θ and rEag1 in synaptosomal and PSD fractions.(A) Subcellular fractionation separated rat brains into multiple fractions: homogenate (H), soluble fraction (S1), crude membrane fraction (P2), synaptosomal fraction (SPM), and two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes), all of which were subject to immunoblotting analyses with the indicated antibodies. 25 µg and 5 µg refer to the amount of total protein loaded in each lane. (B) Quantitative analyses of protein abundance in different subcellular fractions. Densitometric scans of immunoblots were obtained from three to five independent experiments. Data were presented as normalized values with respect to cognate protein expression levels in the homogenate (H) fraction.
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pone-0041203-g006: Localization of 14-3-3θ and rEag1 in synaptosomal and PSD fractions.(A) Subcellular fractionation separated rat brains into multiple fractions: homogenate (H), soluble fraction (S1), crude membrane fraction (P2), synaptosomal fraction (SPM), and two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes), all of which were subject to immunoblotting analyses with the indicated antibodies. 25 µg and 5 µg refer to the amount of total protein loaded in each lane. (B) Quantitative analyses of protein abundance in different subcellular fractions. Densitometric scans of immunoblots were obtained from three to five independent experiments. Data were presented as normalized values with respect to cognate protein expression levels in the homogenate (H) fraction.

Mentions: We also compared the subcellular distribution of 14-3-3θ and rEag1 by performing the subcellular fractionation experiment. Via sucrose gradient centrifugation, the P2 fraction of rat forebrain homogenates was separated into multiple fractions, from which we collected the synaptosomal (SPM) fraction. As illustrated in the left panel of Figure 6A, both 14-3-3θ and rEag1 proteins were present in the SPM fraction, implying a co-localization of the two proteins in the synapse. Extractions with Triton X-100 further divided the SPM fraction into the postsynaptic density I (PSD I; one Triton X-100 wash) and the PSD II (two Triton X-100 washes) fractions, which provide insightful information on the subcellular localization of synapse-related proteins: the postsynaptic marker PSD-95 was highly enriched in both the PSD I and the PSD II fractions, whereas the presynaptic marker synaptophysin was only present in the PSD I fraction (Fig. 6A, right panel). Consistent with the aforementioned punctate co-localization patterns in cultured hippocampal neurons (see Fig. 5B), we found that rEag1 as well as 14-3-3θ were detected in both the PSD I and the PSD II fractions (Fig. 6A, right panel). Figure 6B provides a quantitative summary of the relative abundance of each protein in different subcellular fractions. Consistent with the co-immunoprecipitation result (see Fig. 4A), rEag1, but not rEag2, displays a similar synaptic localization profile to that of 14-3-3θ. Altogether these data provide strong evidence in support of the association of 14-3-3θ with rEag1 in the brain.


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)

Localization of 14-3-3θ and rEag1 in synaptosomal and PSD fractions.(A) Subcellular fractionation separated rat brains into multiple fractions: homogenate (H), soluble fraction (S1), crude membrane fraction (P2), synaptosomal fraction (SPM), and two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes), all of which were subject to immunoblotting analyses with the indicated antibodies. 25 µg and 5 µg refer to the amount of total protein loaded in each lane. (B) Quantitative analyses of protein abundance in different subcellular fractions. Densitometric scans of immunoblots were obtained from three to five independent experiments. Data were presented as normalized values with respect to cognate protein expression levels in the homogenate (H) fraction.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0041203-g006: Localization of 14-3-3θ and rEag1 in synaptosomal and PSD fractions.(A) Subcellular fractionation separated rat brains into multiple fractions: homogenate (H), soluble fraction (S1), crude membrane fraction (P2), synaptosomal fraction (SPM), and two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes), all of which were subject to immunoblotting analyses with the indicated antibodies. 25 µg and 5 µg refer to the amount of total protein loaded in each lane. (B) Quantitative analyses of protein abundance in different subcellular fractions. Densitometric scans of immunoblots were obtained from three to five independent experiments. Data were presented as normalized values with respect to cognate protein expression levels in the homogenate (H) fraction.
Mentions: We also compared the subcellular distribution of 14-3-3θ and rEag1 by performing the subcellular fractionation experiment. Via sucrose gradient centrifugation, the P2 fraction of rat forebrain homogenates was separated into multiple fractions, from which we collected the synaptosomal (SPM) fraction. As illustrated in the left panel of Figure 6A, both 14-3-3θ and rEag1 proteins were present in the SPM fraction, implying a co-localization of the two proteins in the synapse. Extractions with Triton X-100 further divided the SPM fraction into the postsynaptic density I (PSD I; one Triton X-100 wash) and the PSD II (two Triton X-100 washes) fractions, which provide insightful information on the subcellular localization of synapse-related proteins: the postsynaptic marker PSD-95 was highly enriched in both the PSD I and the PSD II fractions, whereas the presynaptic marker synaptophysin was only present in the PSD I fraction (Fig. 6A, right panel). Consistent with the aforementioned punctate co-localization patterns in cultured hippocampal neurons (see Fig. 5B), we found that rEag1 as well as 14-3-3θ were detected in both the PSD I and the PSD II fractions (Fig. 6A, right panel). Figure 6B provides a quantitative summary of the relative abundance of each protein in different subcellular fractions. Consistent with the co-immunoprecipitation result (see Fig. 4A), rEag1, but not rEag2, displays a similar synaptic localization profile to that of 14-3-3θ. Altogether these data provide strong evidence in support of the association of 14-3-3θ with rEag1 in the brain.

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