Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of rEag1-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag1-II, rEag1-III, and rEag1-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag1 constructs. (C) Membrane localization of the GFP-rEag1 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag1 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag1 chimeric channels. Note the presence of rEag2-like GFP puncta density in rEag1-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
getmorefigures.php?uid=PMC3926332&req=5

Figure 5: Characterization of rEag1-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag1-II, rEag1-III, and rEag1-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag1 constructs. (C) Membrane localization of the GFP-rEag1 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag1 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag1 chimeric channels. Note the presence of rEag2-like GFP puncta density in rEag1-II only. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)

Mentions: To further examine whether the post-CNBHD region may contribute to the differential subcellular localization of the two Eag isoforms, we generated three additional rEag1 chimeras, namely rEag1-II, rEag1-III, and rEag1-IV, each of which contains a separate rEag2 post-CNBHD segment with a distinct sequence divergence from that of rEag1 (Figure 5A). In the heterologous expression system, the functional and membrane trafficking properties of all three rEag1 chimeras were similar to those of wild-type rEag1 (Figure 5B-C). We then inspected the subcellular localization of these GFP-tagged chimeras in DIV12 hippocampal neurons (Figure 5D) (see also Additional file 2). A quantitative analysis of the GFP fluorescence results indicated that the GFP puncta densities of GFP-rEag1-II, GFP-rEag1-III, and GFP-rEag1-IV chimeras were about 22 ± 8, 124 ± 15, and 108 ± 11, respectively (Figure 5E). In other words, a rEag2-like pattern with very few GFP puncta was only observed in the GFP-rEag1-II chimera, which is the protein where the proximal post-CNBHD region (rEag1 A723-R807) has been substituted with its rEag2 counterpart (rEag2 L719-G808).


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 rEag1-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag1-II, rEag1-III, and rEag1-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag1 constructs. (C) Membrane localization of the GFP-rEag1 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag1 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag1 chimeric channels. Note the presence of rEag2-like GFP puncta density in rEag1-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 5: Characterization of rEag1-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag1-II, rEag1-III, and rEag1-IV chimeras. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated rEag1 constructs. (C) Membrane localization of the GFP-rEag1 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag1 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag1 chimeric channels. Note the presence of rEag2-like GFP puncta density in rEag1-II only. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)
Mentions: To further examine whether the post-CNBHD region may contribute to the differential subcellular localization of the two Eag isoforms, we generated three additional rEag1 chimeras, namely rEag1-II, rEag1-III, and rEag1-IV, each of which contains a separate rEag2 post-CNBHD segment with a distinct sequence divergence from that of rEag1 (Figure 5A). In the heterologous expression system, the functional and membrane trafficking properties of all three rEag1 chimeras were similar to those of wild-type rEag1 (Figure 5B-C). We then inspected the subcellular localization of these GFP-tagged chimeras in DIV12 hippocampal neurons (Figure 5D) (see also Additional file 2). A quantitative analysis of the GFP fluorescence results indicated that the GFP puncta densities of GFP-rEag1-II, GFP-rEag1-III, and GFP-rEag1-IV chimeras were about 22 ± 8, 124 ± 15, and 108 ± 11, respectively (Figure 5E). In other words, a rEag2-like pattern with very few GFP puncta was only observed in the GFP-rEag1-II chimera, which is the protein where the proximal post-CNBHD region (rEag1 A723-R807) has been substituted with its rEag2 counterpart (rEag2 L719-G808).

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