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The punctate localization of rat Eag1 K+ channels is conferred by the proximal post-CNBHD region.

Chuang CC, Jow GM, Lin HM, Weng YH, Hu JH, Peng YJ, Chiu YC, Chiu MM, Jeng CJ - BMC Neurosci (2014)

Bottom Line: Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95.Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels.Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, No, 155, Section 2, Li-Non Street, Taipei 12212, Taiwan. cjjeng@ym.edu.tw.

ABSTRACT

Background: In mammals, Eag K+ channels (KV10) are exclusively expressed in the brain and comprise two isoforms: Eag1 (KV10.1) and Eag2 (KV10.2). Despite their wide presence in various regions of the brain, the functional role of Eag K+ channels remains obscure. Here we address this question by characterizing the subcellular localization of rat Eag1 (rEag1) and rat Eag2 (rEag2) in hippocampal neurons, as well as determining the structural basis underlying their different localization patterns.

Results: Immunofluorescence analysis of young and mature hippocampal neurons in culture revealed that endogenous rEag1 and rEag2 K+ channels were present in both the dendrosomatic and the axonal compartments. Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95. Subcellular fractionation analysis further demonstrated a distinct enrichment of rEag1 in the synaptosomal fraction. Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels. To identify the protein region dictating the Eag channel subcellular localization pattern, we generated a variety of different chimeric constructs between rEag1 and rEag2. Quantitative studies of neurons over-expressing these GFP-tagged chimeras indicated that punctate localization was conferred by a segment (A723-R807) within the proximal post-cyclic nucleotide-binding homology domain (post-CNBHD) region in the rEag1 carboxyl terminus.

Conclusions: Our findings suggest that Eag1 and Eag2 K+ channels may modulate membrane excitability in both the dendrosomatic and the axonal compartments and that Eag1 may additionally regulate neurotransmitter release and postsynaptic signaling. Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.

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Synaptic localization of rEag1 channels. (A) Hippocampal neurons were double-stained for rEag1/rEag2 (left panels) and the postsynaptic density marker PSD-95 (middle panels). Scale bar, 25 μm. (B) Quantification of the number of puncta per 100-μm neurite (puncta/100 μm) for PSD-95, rEag1, and rEag2. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from PSD-95. Data were collected from 7-11 different neurons. (C) Quantification of the co-localization of PSD-95 with rEag1 or rEag2. The data illustrate the fraction of PSD-95 puncta that were co-localized with rEag1/2 puncta, as well as the fraction of rEag1/2 puncta that were co-localized with PSD-95 puncta. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from rEag1. Data were collected from 7-8 different neurons. (D) Subcellular fractionation of rat brains: the homogenate (H), the soluble fraction (S1), the crude membrane fraction (P2), the synaptosomal fraction (SPM), and the two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes). The left panel (25 μg) illustrates the primary fractionation profile, whereas the right panel (5 μg) exemplifies the further enrichment pattern in the three sub-fractions of synaptosomes. All fractions 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.
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Figure 2: Synaptic localization of rEag1 channels. (A) Hippocampal neurons were double-stained for rEag1/rEag2 (left panels) and the postsynaptic density marker PSD-95 (middle panels). Scale bar, 25 μm. (B) Quantification of the number of puncta per 100-μm neurite (puncta/100 μm) for PSD-95, rEag1, and rEag2. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from PSD-95. Data were collected from 7-11 different neurons. (C) Quantification of the co-localization of PSD-95 with rEag1 or rEag2. The data illustrate the fraction of PSD-95 puncta that were co-localized with rEag1/2 puncta, as well as the fraction of rEag1/2 puncta that were co-localized with PSD-95 puncta. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from rEag1. Data were collected from 7-8 different neurons. (D) Subcellular fractionation of rat brains: the homogenate (H), the soluble fraction (S1), the crude membrane fraction (P2), the synaptosomal fraction (SPM), and the two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes). The left panel (25 μg) illustrates the primary fractionation profile, whereas the right panel (5 μg) exemplifies the further enrichment pattern in the three sub-fractions of synaptosomes. All fractions 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.

Mentions: As mentioned above, the punctate localization of rEag1 channels was copious in DIV12 neurons, which are known to form numerous and widespread synaptic connections. We quantified the rEag1 puncta within the neurons by calculating the puncta density, which was defined as the number of immunofluorescence puncta per 100-μm neurite (see Methods for detail). The puncta density of rEag1 was about 39 ± 1 (mean ± SEM), which is quite similar to that of the postsynaptic density (PSD) marker PSD-95 (about 42 ± 1) (Figure 2A-B). In contrast, the puncta density of rEag2 was only about 5 ± 1 (Figure 2B). This lack of punctate staining pattern seems to imply that rEag2 is not significantly present at synapses. Consistent with this notion, only about 1 ± 1% of PSD-95 puncta were found to be co-localized with rEag2 puncta, and conversely the PSD-95 co-localization ratio of rEag2 puncta was only about 6 ± 3% (Figure 2C). This contrasts with about 68 ± 2% of PSD-95 puncta being co-localized with rEag1 puncta, and with about 74 ± 2% of rEag1 puncta being co-localized with PSD-95 puncta (Figure 2C).


The punctate localization of rat Eag1 K+ channels is conferred by the proximal post-CNBHD region.

Chuang CC, Jow GM, Lin HM, Weng YH, Hu JH, Peng YJ, Chiu YC, Chiu MM, Jeng CJ - BMC Neurosci (2014)

Synaptic localization of rEag1 channels. (A) Hippocampal neurons were double-stained for rEag1/rEag2 (left panels) and the postsynaptic density marker PSD-95 (middle panels). Scale bar, 25 μm. (B) Quantification of the number of puncta per 100-μm neurite (puncta/100 μm) for PSD-95, rEag1, and rEag2. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from PSD-95. Data were collected from 7-11 different neurons. (C) Quantification of the co-localization of PSD-95 with rEag1 or rEag2. The data illustrate the fraction of PSD-95 puncta that were co-localized with rEag1/2 puncta, as well as the fraction of rEag1/2 puncta that were co-localized with PSD-95 puncta. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from rEag1. Data were collected from 7-8 different neurons. (D) Subcellular fractionation of rat brains: the homogenate (H), the soluble fraction (S1), the crude membrane fraction (P2), the synaptosomal fraction (SPM), and the two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes). The left panel (25 μg) illustrates the primary fractionation profile, whereas the right panel (5 μg) exemplifies the further enrichment pattern in the three sub-fractions of synaptosomes. All fractions 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926332&req=5

Figure 2: Synaptic localization of rEag1 channels. (A) Hippocampal neurons were double-stained for rEag1/rEag2 (left panels) and the postsynaptic density marker PSD-95 (middle panels). Scale bar, 25 μm. (B) Quantification of the number of puncta per 100-μm neurite (puncta/100 μm) for PSD-95, rEag1, and rEag2. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from PSD-95. Data were collected from 7-11 different neurons. (C) Quantification of the co-localization of PSD-95 with rEag1 or rEag2. The data illustrate the fraction of PSD-95 puncta that were co-localized with rEag1/2 puncta, as well as the fraction of rEag1/2 puncta that were co-localized with PSD-95 puncta. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from rEag1. Data were collected from 7-8 different neurons. (D) Subcellular fractionation of rat brains: the homogenate (H), the soluble fraction (S1), the crude membrane fraction (P2), the synaptosomal fraction (SPM), and the two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes). The left panel (25 μg) illustrates the primary fractionation profile, whereas the right panel (5 μg) exemplifies the further enrichment pattern in the three sub-fractions of synaptosomes. All fractions 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.
Mentions: As mentioned above, the punctate localization of rEag1 channels was copious in DIV12 neurons, which are known to form numerous and widespread synaptic connections. We quantified the rEag1 puncta within the neurons by calculating the puncta density, which was defined as the number of immunofluorescence puncta per 100-μm neurite (see Methods for detail). The puncta density of rEag1 was about 39 ± 1 (mean ± SEM), which is quite similar to that of the postsynaptic density (PSD) marker PSD-95 (about 42 ± 1) (Figure 2A-B). In contrast, the puncta density of rEag2 was only about 5 ± 1 (Figure 2B). This lack of punctate staining pattern seems to imply that rEag2 is not significantly present at synapses. Consistent with this notion, only about 1 ± 1% of PSD-95 puncta were found to be co-localized with rEag2 puncta, and conversely the PSD-95 co-localization ratio of rEag2 puncta was only about 6 ± 3% (Figure 2C). This contrasts with about 68 ± 2% of PSD-95 puncta being co-localized with rEag1 puncta, and with about 74 ± 2% of rEag1 puncta being co-localized with PSD-95 puncta (Figure 2C).

Bottom Line: Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95.Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels.Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, No, 155, Section 2, Li-Non Street, Taipei 12212, Taiwan. cjjeng@ym.edu.tw.

ABSTRACT

Background: In mammals, Eag K+ channels (KV10) are exclusively expressed in the brain and comprise two isoforms: Eag1 (KV10.1) and Eag2 (KV10.2). Despite their wide presence in various regions of the brain, the functional role of Eag K+ channels remains obscure. Here we address this question by characterizing the subcellular localization of rat Eag1 (rEag1) and rat Eag2 (rEag2) in hippocampal neurons, as well as determining the structural basis underlying their different localization patterns.

Results: Immunofluorescence analysis of young and mature hippocampal neurons in culture revealed that endogenous rEag1 and rEag2 K+ channels were present in both the dendrosomatic and the axonal compartments. Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95. Subcellular fractionation analysis further demonstrated a distinct enrichment of rEag1 in the synaptosomal fraction. Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels. To identify the protein region dictating the Eag channel subcellular localization pattern, we generated a variety of different chimeric constructs between rEag1 and rEag2. Quantitative studies of neurons over-expressing these GFP-tagged chimeras indicated that punctate localization was conferred by a segment (A723-R807) within the proximal post-cyclic nucleotide-binding homology domain (post-CNBHD) region in the rEag1 carboxyl terminus.

Conclusions: Our findings suggest that Eag1 and Eag2 K+ channels may modulate membrane excitability in both the dendrosomatic and the axonal compartments and that Eag1 may additionally regulate neurotransmitter release and postsynaptic signaling. Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.

Show MeSH
Related in: MedlinePlus