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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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Characterization of rEag2-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag2-II, rEag2-III, and rEag2-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag2 constructs. (C) Membrane localization of the GFP-rEag2 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag2 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag2 chimeric channels. Note the presence of rEag1-like GFP puncta density in rEag2-II only. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)
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Figure 6: Characterization of rEag2-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag2-II, rEag2-III, and rEag2-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag2 constructs. (C) Membrane localization of the GFP-rEag2 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag2 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag2 chimeric channels. Note the presence of rEag1-like GFP puncta density in rEag2-II only. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)

Mentions: The above findings imply that the proximal post-CNBHD region of rEag1 is likely to play an essential role in the expression of a punctate localization pattern. To further test this hypothesis, we then constructed three reverse rEag2 chimeras, namely rEag2-II, rEag2-III, and rEag2-IV, each of which harbored a segment of the rEag1 post-CNBHD sequences (Figure 6A). In the heterologous expression system, the three rEag2 chimeras were similar to each other in terms of their functional and membrane trafficking properties (Figure 6B-C). Figure 6D (see also Additional file 3) illustrates representative localization patterns of the GFP-tagged chimeras in hippocampal neurons. The calculated values of GFP puncta densities were about 86 ± 6 (GFP-rEag2-II), 13 ± 4 (GFP-rEag2-III), and 13 ± 3 (GFP-rEag2-IV) (Figure 6E). Therefore, prominent punctate localization was only found when GFP-rEag2-II, the chimera containing the rEag1 segment A723-R807, was present.


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)

Characterization of rEag2-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag2-II, rEag2-III, and rEag2-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag2 constructs. (C) Membrane localization of the GFP-rEag2 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag2 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag2 chimeric channels. Note the presence of rEag1-like GFP puncta density in rEag2-II only. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Characterization of rEag2-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag2-II, rEag2-III, and rEag2-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag2 constructs. (C) Membrane localization of the GFP-rEag2 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag2 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag2 chimeric channels. Note the presence of rEag1-like GFP puncta density in rEag2-II only. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)
Mentions: The above findings imply that the proximal post-CNBHD region of rEag1 is likely to play an essential role in the expression of a punctate localization pattern. To further test this hypothesis, we then constructed three reverse rEag2 chimeras, namely rEag2-II, rEag2-III, and rEag2-IV, each of which harbored a segment of the rEag1 post-CNBHD sequences (Figure 6A). In the heterologous expression system, the three rEag2 chimeras were similar to each other in terms of their functional and membrane trafficking properties (Figure 6B-C). Figure 6D (see also Additional file 3) illustrates representative localization patterns of the GFP-tagged chimeras in hippocampal neurons. The calculated values of GFP puncta densities were about 86 ± 6 (GFP-rEag2-II), 13 ± 4 (GFP-rEag2-III), and 13 ± 3 (GFP-rEag2-IV) (Figure 6E). Therefore, prominent punctate localization was only found when GFP-rEag2-II, the chimera containing the rEag1 segment A723-R807, was present.

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