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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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The effect of 14-3-3θ over-expression on rEag1 K+ currents.(A) (Left panel) Representative K+ currents recorded from HEK293T cells expressing rEag1 in the absence or presence of 14-3-3θ. HEK293T cells were co-transfected with the cDNAs for rEag1 and myc-vector or myc-14-3-3θ in the molar ratio of 1∶5. The holding potential was −90 mV. The pulse protocol comprised 300-ms depolarizing test pulses ranging from −90 to +50 mV, with 10-mV increments. (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of myc-14-3-3θ. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1 control (*, t-test: p<0.05). (B) (Left panel) Representative K+ currents recorded from oocytes expressing rEag1 in the absence or presence of 14-3-3θ. The molar ratio for cRNA co-injection was 1∶5 and 1∶10 for 14-3-3θ and Kvβ1, respectively. The pulse protocol was identical to that described in (A). (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of 14-3-3θ. (C) Biophysical properties of rEag1 channels in the absence (open circles) or presence (filled diamonds) of 14-3-3θ. The voltage-dependant curves for steady-state activation (upper left panel), activation kinetics (upper right panel), deactivation kinetics (lower left panel), and non-superimposable Cole-Moore shift (lower right panel) were analyzed as described previously [17]. Data were collected from recordings performed in oocytes.
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pone-0041203-g007: The effect of 14-3-3θ over-expression on rEag1 K+ currents.(A) (Left panel) Representative K+ currents recorded from HEK293T cells expressing rEag1 in the absence or presence of 14-3-3θ. HEK293T cells were co-transfected with the cDNAs for rEag1 and myc-vector or myc-14-3-3θ in the molar ratio of 1∶5. The holding potential was −90 mV. The pulse protocol comprised 300-ms depolarizing test pulses ranging from −90 to +50 mV, with 10-mV increments. (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of myc-14-3-3θ. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1 control (*, t-test: p<0.05). (B) (Left panel) Representative K+ currents recorded from oocytes expressing rEag1 in the absence or presence of 14-3-3θ. The molar ratio for cRNA co-injection was 1∶5 and 1∶10 for 14-3-3θ and Kvβ1, respectively. The pulse protocol was identical to that described in (A). (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of 14-3-3θ. (C) Biophysical properties of rEag1 channels in the absence (open circles) or presence (filled diamonds) of 14-3-3θ. The voltage-dependant curves for steady-state activation (upper left panel), activation kinetics (upper right panel), deactivation kinetics (lower left panel), and non-superimposable Cole-Moore shift (lower right panel) were analyzed as described previously [17]. Data were collected from recordings performed in oocytes.

Mentions: If the preceding inference on the protein-protein interaction is true, then is it possible that 14-3-3θ may affect the functional property of rEag1 K+ channels? To address this question, we studied the functional expression of rEag1 channels in the absence or presence of the over-expression of 14-3-3θ. Figure 7A exemplifies the representative result observed in HEK293T cells: over-expression of 14-3-3θ led to about 30% reduction of the amplitude of rEag1 K+ currents. Similarly, in Xenopus oocytes, over-expression of 14-3-3θ resulted in more than 50% reduction of rEag1 K+ currents (Fig. 7B). By contrast, the functional expression of rEag1 channels was not significantly affected by the over-expression of the auxiliary β1 subunit of Kv channels (Kvβ1) (Fig. 7B), a protein similar in size with 14-3-3θ. Other than the suppression of current amplitude, co-expression with 14-3-3θ failed to exert discernible effect on the gating properties (such as steady-state voltage dependence and gating kinetics) of rEag1 channels (Fig. 7C).


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)

The effect of 14-3-3θ over-expression on rEag1 K+ currents.(A) (Left panel) Representative K+ currents recorded from HEK293T cells expressing rEag1 in the absence or presence of 14-3-3θ. HEK293T cells were co-transfected with the cDNAs for rEag1 and myc-vector or myc-14-3-3θ in the molar ratio of 1∶5. The holding potential was −90 mV. The pulse protocol comprised 300-ms depolarizing test pulses ranging from −90 to +50 mV, with 10-mV increments. (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of myc-14-3-3θ. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1 control (*, t-test: p<0.05). (B) (Left panel) Representative K+ currents recorded from oocytes expressing rEag1 in the absence or presence of 14-3-3θ. The molar ratio for cRNA co-injection was 1∶5 and 1∶10 for 14-3-3θ and Kvβ1, respectively. The pulse protocol was identical to that described in (A). (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of 14-3-3θ. (C) Biophysical properties of rEag1 channels in the absence (open circles) or presence (filled diamonds) of 14-3-3θ. The voltage-dependant curves for steady-state activation (upper left panel), activation kinetics (upper right panel), deactivation kinetics (lower left panel), and non-superimposable Cole-Moore shift (lower right panel) were analyzed as described previously [17]. Data were collected from recordings performed in oocytes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401112&req=5

pone-0041203-g007: The effect of 14-3-3θ over-expression on rEag1 K+ currents.(A) (Left panel) Representative K+ currents recorded from HEK293T cells expressing rEag1 in the absence or presence of 14-3-3θ. HEK293T cells were co-transfected with the cDNAs for rEag1 and myc-vector or myc-14-3-3θ in the molar ratio of 1∶5. The holding potential was −90 mV. The pulse protocol comprised 300-ms depolarizing test pulses ranging from −90 to +50 mV, with 10-mV increments. (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of myc-14-3-3θ. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1 control (*, t-test: p<0.05). (B) (Left panel) Representative K+ currents recorded from oocytes expressing rEag1 in the absence or presence of 14-3-3θ. The molar ratio for cRNA co-injection was 1∶5 and 1∶10 for 14-3-3θ and Kvβ1, respectively. The pulse protocol was identical to that described in (A). (Right panel) Normalized mean K+ current density (at +40 mV) of rEag1 channels in the absence or presence of 14-3-3θ. (C) Biophysical properties of rEag1 channels in the absence (open circles) or presence (filled diamonds) of 14-3-3θ. The voltage-dependant curves for steady-state activation (upper left panel), activation kinetics (upper right panel), deactivation kinetics (lower left panel), and non-superimposable Cole-Moore shift (lower right panel) were analyzed as described previously [17]. Data were collected from recordings performed in oocytes.
Mentions: If the preceding inference on the protein-protein interaction is true, then is it possible that 14-3-3θ may affect the functional property of rEag1 K+ channels? To address this question, we studied the functional expression of rEag1 channels in the absence or presence of the over-expression of 14-3-3θ. Figure 7A exemplifies the representative result observed in HEK293T cells: over-expression of 14-3-3θ led to about 30% reduction of the amplitude of rEag1 K+ currents. Similarly, in Xenopus oocytes, over-expression of 14-3-3θ resulted in more than 50% reduction of rEag1 K+ currents (Fig. 7B). By contrast, the functional expression of rEag1 channels was not significantly affected by the over-expression of the auxiliary β1 subunit of Kv channels (Kvβ1) (Fig. 7B), a protein similar in size with 14-3-3θ. Other than the suppression of current amplitude, co-expression with 14-3-3θ failed to exert discernible effect on the gating properties (such as steady-state voltage dependence and gating kinetics) of rEag1 channels (Fig. 7C).

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