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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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Expression of GFP-rEag1-K848X channels in hippocampal neurons. (Left panel) GFP-rEag1-K848X was over-expressed in DIV12 hippocampal neurons. Similar to GFP-rEag1 channels, the GFP signal arising from the truncation mutant also displayed the characteristic punctate pattern. Scale bar, 25 μm. (Right panel) Quantification of the number of GFP puncta per neuron for rEag1-K848X. (*: 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 7: Expression of GFP-rEag1-K848X channels in hippocampal neurons. (Left panel) GFP-rEag1-K848X was over-expressed in DIV12 hippocampal neurons. Similar to GFP-rEag1 channels, the GFP signal arising from the truncation mutant also displayed the characteristic punctate pattern. Scale bar, 25 μm. (Right panel) Quantification of the number of GFP puncta per neuron for rEag1-K848X. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)

Mentions: The foregoing observations directly imply that the distal post-CNBHD region, including the carboxyl assembly domain (CAD) (Figure 3C), is not involved in determining the subcellular localization of rEag1. To address this issue, we focused on a previously identified truncation mutant that lacks CAD, K848X, the membrane trafficking and biophysical properties of which are similar to those of wild-type rEag1 [18]. Figure 7 (see also Additional file 4) shows that GFP-rEag1-K848X does indeed display considerable punctate localization in DIV12 hippocampal neurons. The GFP puncta density of GFP-rEag1-K848X (104 ± 15), while less than that of GFP-rEag1, is about 6-fold higher than that of GFP-rEag2, which is consistent with the idea that the distal post-CNBHD region is not required for conferring the punctate localization on rEag1 channels.


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)

Expression of GFP-rEag1-K848X channels in hippocampal neurons. (Left panel) GFP-rEag1-K848X was over-expressed in DIV12 hippocampal neurons. Similar to GFP-rEag1 channels, the GFP signal arising from the truncation mutant also displayed the characteristic punctate pattern. Scale bar, 25 μm. (Right panel) Quantification of the number of GFP puncta per neuron for rEag1-K848X. (*: 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 7: Expression of GFP-rEag1-K848X channels in hippocampal neurons. (Left panel) GFP-rEag1-K848X was over-expressed in DIV12 hippocampal neurons. Similar to GFP-rEag1 channels, the GFP signal arising from the truncation mutant also displayed the characteristic punctate pattern. Scale bar, 25 μm. (Right panel) Quantification of the number of GFP puncta per neuron for rEag1-K848X. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)
Mentions: The foregoing observations directly imply that the distal post-CNBHD region, including the carboxyl assembly domain (CAD) (Figure 3C), is not involved in determining the subcellular localization of rEag1. To address this issue, we focused on a previously identified truncation mutant that lacks CAD, K848X, the membrane trafficking and biophysical properties of which are similar to those of wild-type rEag1 [18]. Figure 7 (see also Additional file 4) shows that GFP-rEag1-K848X does indeed display considerable punctate localization in DIV12 hippocampal neurons. The GFP puncta density of GFP-rEag1-K848X (104 ± 15), while less than that of GFP-rEag1, is about 6-fold higher than that of GFP-rEag2, which is consistent with the idea that the distal post-CNBHD region is not required for conferring the punctate localization on rEag1 channels.

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