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

Subcellular localization of native rEag1 and rEag2 channels in young and mature hippocampal neurons in culture. Dissociated hippocampal neurons at DIV3, DIV7, and DIV12 were immunostained with the anti-rEag1 (A, B) or the anti-rEag2 (C, D) antibodies (shown in green; left panels), followed by counterstaining with the antibody for the dendritic marker MAP2 or the axonal marker tau (shown in red; middle panels). Merged images are shown in the right panels. (A) rEag1 immunoreactivities were localized in cell bodies, as well as in MAP2-positive and MAP2-negative (arrows) processes. (B) rEag1 immunoreactivities were present in the axonal compartment that was clearly defined by the immunofluorescence signal of tau (arrows). For DIV12 neurons in both (A) and (B), note the presence of punctate rEag1 staining patterns throughout proximal and distal neurites. (C, D) rEag2 immunoreactivities were present in both MAP2-positive and tau-positive processes. No significant rEag2 puncta were observed. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Subcellular localization of native rEag1 and rEag2 channels in young and mature hippocampal neurons in culture. Dissociated hippocampal neurons at DIV3, DIV7, and DIV12 were immunostained with the anti-rEag1 (A, B) or the anti-rEag2 (C, D) antibodies (shown in green; left panels), followed by counterstaining with the antibody for the dendritic marker MAP2 or the axonal marker tau (shown in red; middle panels). Merged images are shown in the right panels. (A) rEag1 immunoreactivities were localized in cell bodies, as well as in MAP2-positive and MAP2-negative (arrows) processes. (B) rEag1 immunoreactivities were present in the axonal compartment that was clearly defined by the immunofluorescence signal of tau (arrows). For DIV12 neurons in both (A) and (B), note the presence of punctate rEag1 staining patterns throughout proximal and distal neurites. (C, D) rEag2 immunoreactivities were present in both MAP2-positive and tau-positive processes. No significant rEag2 puncta were observed. Scale bar, 25 μm.

Mentions: We began by asking whether rEag1 and rEag2 K+ channels are present in axons. For isolated hippocampal neurons, the process of axon-dendrite polarization initiates within the first 36 hours in culture and by 72 hours in culture, a single neurite undergoing fast elongation has become an axon [19]. We therefore decided to perform immunofluorescence characterization in DIV3, DIV7, and DIV12 neurons. As illustrated in Figure 1A, rEag1 staining was found to co-localize with the dendrite marker microtubule-associated protein 2 (MAP2) in all three populations of cultured hippocampal neurons, which is consistent with our previous observation that rEag1 K+ channels are present in the dendrosomatic compartment. Furthermore, the characteristic punctate localization of rEag1 channels was visible in DIV7 neurons and was profusely present in DIV12 neurons. Interestingly, by closely inspecting the DIV3 and DIV7 neurons, whose neurite networks were less sophisticated, we also observed significant rEag1 immunofluorescence signal in MAP2-negative neurites, which implies that rEag1 channels may be present in axons as well. We then compared the immunofluorescence signals of rEag1 with those of the axon marker tau. Figure 1B shows that in DIV3 neurons, where the location of the axon is clearly defined by the tau immunofluorescence signal, rEag1 channels are present within the soma and also in the complete axonal compartment. A similar tau-positive rEag1 immunostaining pattern can be observed in DIV7 and DIV12 neurons as well, although a significant fraction of the immunofluorescence signal may represent the presence of the channel protein in axons stemming from neighboring neurons. These findings thus demonstrate that rEag1 channels are universally distributed in both the dendrosomatic and the axonal compartments of hippocampal neurons.


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)

Subcellular localization of native rEag1 and rEag2 channels in young and mature hippocampal neurons in culture. Dissociated hippocampal neurons at DIV3, DIV7, and DIV12 were immunostained with the anti-rEag1 (A, B) or the anti-rEag2 (C, D) antibodies (shown in green; left panels), followed by counterstaining with the antibody for the dendritic marker MAP2 or the axonal marker tau (shown in red; middle panels). Merged images are shown in the right panels. (A) rEag1 immunoreactivities were localized in cell bodies, as well as in MAP2-positive and MAP2-negative (arrows) processes. (B) rEag1 immunoreactivities were present in the axonal compartment that was clearly defined by the immunofluorescence signal of tau (arrows). For DIV12 neurons in both (A) and (B), note the presence of punctate rEag1 staining patterns throughout proximal and distal neurites. (C, D) rEag2 immunoreactivities were present in both MAP2-positive and tau-positive processes. No significant rEag2 puncta were observed. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Subcellular localization of native rEag1 and rEag2 channels in young and mature hippocampal neurons in culture. Dissociated hippocampal neurons at DIV3, DIV7, and DIV12 were immunostained with the anti-rEag1 (A, B) or the anti-rEag2 (C, D) antibodies (shown in green; left panels), followed by counterstaining with the antibody for the dendritic marker MAP2 or the axonal marker tau (shown in red; middle panels). Merged images are shown in the right panels. (A) rEag1 immunoreactivities were localized in cell bodies, as well as in MAP2-positive and MAP2-negative (arrows) processes. (B) rEag1 immunoreactivities were present in the axonal compartment that was clearly defined by the immunofluorescence signal of tau (arrows). For DIV12 neurons in both (A) and (B), note the presence of punctate rEag1 staining patterns throughout proximal and distal neurites. (C, D) rEag2 immunoreactivities were present in both MAP2-positive and tau-positive processes. No significant rEag2 puncta were observed. Scale bar, 25 μm.
Mentions: We began by asking whether rEag1 and rEag2 K+ channels are present in axons. For isolated hippocampal neurons, the process of axon-dendrite polarization initiates within the first 36 hours in culture and by 72 hours in culture, a single neurite undergoing fast elongation has become an axon [19]. We therefore decided to perform immunofluorescence characterization in DIV3, DIV7, and DIV12 neurons. As illustrated in Figure 1A, rEag1 staining was found to co-localize with the dendrite marker microtubule-associated protein 2 (MAP2) in all three populations of cultured hippocampal neurons, which is consistent with our previous observation that rEag1 K+ channels are present in the dendrosomatic compartment. Furthermore, the characteristic punctate localization of rEag1 channels was visible in DIV7 neurons and was profusely present in DIV12 neurons. Interestingly, by closely inspecting the DIV3 and DIV7 neurons, whose neurite networks were less sophisticated, we also observed significant rEag1 immunofluorescence signal in MAP2-negative neurites, which implies that rEag1 channels may be present in axons as well. We then compared the immunofluorescence signals of rEag1 with those of the axon marker tau. Figure 1B shows that in DIV3 neurons, where the location of the axon is clearly defined by the tau immunofluorescence signal, rEag1 channels are present within the soma and also in the complete axonal compartment. A similar tau-positive rEag1 immunostaining pattern can be observed in DIV7 and DIV12 neurons as well, although a significant fraction of the immunofluorescence signal may represent the presence of the channel protein in axons stemming from neighboring neurons. These findings thus demonstrate that rEag1 channels are universally distributed in both the dendrosomatic and the axonal compartments of hippocampal neurons.

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