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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 and GFP-rEag2 channels in HEK293T cells and hippocampal neurons. (A) Immunofluorescence staining and functional expression of GFP-rEag1 and GFP-rEag2 K+ channels in HEK293T cells. GFP fluorescence (shown in green) and rEag1/rEag2 immunofluorescence (shown in red) signals demonstrated lucid co-localization at the membrane region. Scale bar, 10 μm. Whole-cell patch clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +50 mV (rEag1) or from -90 to +30 mV (rEag2), with 10-mV increments. (B) Over-expression of GFP-rEag1/rEag2 in DIV12 hippocampal neurons. GFP signal is shown in green, and MAP2 immunofluorescence signal in red. Note the presence of prominent GFP puncta for rEag1, but not rEag2. Scale bar, 25 μm. (C) (Top) Schematic representation of the structural topology of Eag K+ channel. (Bottom) Protein sequence alignment between rEag1 and rEag2 over the post-CNBHD region. Yellow shade: identical residues. Green shade: homologous residues. Sequence alignment analysis was implemented with the Vector NTI software (InforMax).
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Figure 3: Expression of GFP-rEag1 and GFP-rEag2 channels in HEK293T cells and hippocampal neurons. (A) Immunofluorescence staining and functional expression of GFP-rEag1 and GFP-rEag2 K+ channels in HEK293T cells. GFP fluorescence (shown in green) and rEag1/rEag2 immunofluorescence (shown in red) signals demonstrated lucid co-localization at the membrane region. Scale bar, 10 μm. Whole-cell patch clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +50 mV (rEag1) or from -90 to +30 mV (rEag2), with 10-mV increments. (B) Over-expression of GFP-rEag1/rEag2 in DIV12 hippocampal neurons. GFP signal is shown in green, and MAP2 immunofluorescence signal in red. Note the presence of prominent GFP puncta for rEag1, but not rEag2. Scale bar, 25 μm. (C) (Top) Schematic representation of the structural topology of Eag K+ channel. (Bottom) Protein sequence alignment between rEag1 and rEag2 over the post-CNBHD region. Yellow shade: identical residues. Green shade: homologous residues. Sequence alignment analysis was implemented with the Vector NTI software (InforMax).

Mentions: To further demonstrate that the punctate localization pattern was indeed an innate distinction between the two channel isoforms, we studied next the exogenous over-expression of rEag1 and rEag2 proteins in hippocampal neurons. A GFP-tag was engineered to be attached to the amino (N) terminus of both rEag1 and rEag2. Upon over-expression in HEK293T cells, the GFP-tagged constructs displayed a clear membrane localization pattern and produced functional K+ currents (Figure 3A); these findings indicate that the membrane trafficking and biophysical properties of the GFP-tagged channels are similar to those of their wild-type counterparts. The cDNAs encoding the GFP-tagged proteins were then transfected into DIV7 neurons and this was followed by confocal microscopic analyses at 5 days post-transfection. As shown in Figure 3B (see also Additional file 1), over-expressed GFP-rEag1 channels, but not over-expressed GFP-rEag2 channels, displayed significant punctate localization in DIV12 neurons. These findings are reminiscent of the differential subcellular localization of endogenous rEag1 and rEag2 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 and GFP-rEag2 channels in HEK293T cells and hippocampal neurons. (A) Immunofluorescence staining and functional expression of GFP-rEag1 and GFP-rEag2 K+ channels in HEK293T cells. GFP fluorescence (shown in green) and rEag1/rEag2 immunofluorescence (shown in red) signals demonstrated lucid co-localization at the membrane region. Scale bar, 10 μm. Whole-cell patch clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +50 mV (rEag1) or from -90 to +30 mV (rEag2), with 10-mV increments. (B) Over-expression of GFP-rEag1/rEag2 in DIV12 hippocampal neurons. GFP signal is shown in green, and MAP2 immunofluorescence signal in red. Note the presence of prominent GFP puncta for rEag1, but not rEag2. Scale bar, 25 μm. (C) (Top) Schematic representation of the structural topology of Eag K+ channel. (Bottom) Protein sequence alignment between rEag1 and rEag2 over the post-CNBHD region. Yellow shade: identical residues. Green shade: homologous residues. Sequence alignment analysis was implemented with the Vector NTI software (InforMax).
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Related In: Results  -  Collection

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Figure 3: Expression of GFP-rEag1 and GFP-rEag2 channels in HEK293T cells and hippocampal neurons. (A) Immunofluorescence staining and functional expression of GFP-rEag1 and GFP-rEag2 K+ channels in HEK293T cells. GFP fluorescence (shown in green) and rEag1/rEag2 immunofluorescence (shown in red) signals demonstrated lucid co-localization at the membrane region. Scale bar, 10 μm. Whole-cell patch clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +50 mV (rEag1) or from -90 to +30 mV (rEag2), with 10-mV increments. (B) Over-expression of GFP-rEag1/rEag2 in DIV12 hippocampal neurons. GFP signal is shown in green, and MAP2 immunofluorescence signal in red. Note the presence of prominent GFP puncta for rEag1, but not rEag2. Scale bar, 25 μm. (C) (Top) Schematic representation of the structural topology of Eag K+ channel. (Bottom) Protein sequence alignment between rEag1 and rEag2 over the post-CNBHD region. Yellow shade: identical residues. Green shade: homologous residues. Sequence alignment analysis was implemented with the Vector NTI software (InforMax).
Mentions: To further demonstrate that the punctate localization pattern was indeed an innate distinction between the two channel isoforms, we studied next the exogenous over-expression of rEag1 and rEag2 proteins in hippocampal neurons. A GFP-tag was engineered to be attached to the amino (N) terminus of both rEag1 and rEag2. Upon over-expression in HEK293T cells, the GFP-tagged constructs displayed a clear membrane localization pattern and produced functional K+ currents (Figure 3A); these findings indicate that the membrane trafficking and biophysical properties of the GFP-tagged channels are similar to those of their wild-type counterparts. The cDNAs encoding the GFP-tagged proteins were then transfected into DIV7 neurons and this was followed by confocal microscopic analyses at 5 days post-transfection. As shown in Figure 3B (see also Additional file 1), over-expressed GFP-rEag1 channels, but not over-expressed GFP-rEag2 channels, displayed significant punctate localization in DIV12 neurons. These findings are reminiscent of the differential subcellular localization of endogenous rEag1 and rEag2 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