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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 rEag1-I and rEag2-I chimeric channels. (A) Schematic representation of the construction of rEag1-I and rEag2-I chimeras. For all schematic cartoons hereafter, rEag1 and rEag2 sequences are shown in red and black, respectively. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated Eag constructs. Two-electrode voltage 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 +60 mV (rEag1) or from -100 to +40 mV (rEag2), with 10-mV increments. (C) Membrane localization of GFP-rEag1-I/rEag2-I channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of GFP-rEag1-I/rEag2-I channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for GFP-rEag1, GFP-rEag1-I, GFP-rEag2-I, and GFP-rEag2. The number in parenthesis denotes the amount of neurons analyzed. (*: 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 4: Characterization of rEag1-I and rEag2-I chimeric channels. (A) Schematic representation of the construction of rEag1-I and rEag2-I chimeras. For all schematic cartoons hereafter, rEag1 and rEag2 sequences are shown in red and black, respectively. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated Eag constructs. Two-electrode voltage 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 +60 mV (rEag1) or from -100 to +40 mV (rEag2), with 10-mV increments. (C) Membrane localization of GFP-rEag1-I/rEag2-I channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of GFP-rEag1-I/rEag2-I channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for GFP-rEag1, GFP-rEag1-I, GFP-rEag2-I, and GFP-rEag2. The number in parenthesis denotes the amount of neurons analyzed. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)

Mentions: Both endogenous and over-expressed rEag1 channels displayed a punctate localization, which strongly suggests that one or more specific sequence motifs within its amino acid sequence may govern the subcellular distribution of the K+ channel in neurons. Although rEag1 and rEag2 share about 70% identity in amino acid sequence [4,6], a significant sequence divergence is present within the C-terminal post-CNBHD region, especially from residues A723 through S962 in rEag1 vs. L719 through F988 in rEag2 (Figure 3C). To test the hypothesis that a key structural domain within the post-CNBHD region may contribute to the punctate localization of rEag1 K+ channels, we generated two chimeric constructs (chimera rEag1-I and chimera rEag2-I) by exchanging the majority of the divergent post-CNBHD sequences between the two channel isoforms (Figure 4A). Upon over-expression in HEK293T cells or Xenopus oocytes, both of the chimeric channels yielded significant K+ currents and effective membrane surface expression (Figure 4B-C). We then over-expressed these GFP-tagged chimeras in hippocampal neurons in order to study their subcellular localization patterns. Confocal microscopic analysis revealed that the GFP-rEag1-I chimera displayed a rEag2-like pattern with only a few GFP puncta (Figure 4D) (see also Additional file 2). By way of contrast, the GFP fluorescence of the reverse chimera GFP-rEag2-I exhibited rEag1-like punctate localization (Figure 4D) (see also Additional file 3). We also quantified the puncta density of the GFP signal by calculating the number of GFP puncta per neuron. The GFP puncta densities of GFP-rEag1 and GFP-rEag2 were about 153 ± 5 and 17 ± 2, respectively (Figure 4E). This 9-fold difference in GFP puncta density between over-expressed GFP-rEag1 and GFP-rEag2 channels is almost equivalent to the previously measured 8-fold difference in puncta density between endogenous rEag1 and rEag2 proteins (see Figure 2B). The GFP puncta density of the GFP-rEag1-I chimera was dramatically reduced to about 31 ± 7, which is statistically similar to that of GFP-rEag2 (Figure 4E). In contrast, the GFP puncta density of the GFP-rEag2-I chimera was remarkably increased to about 109 ± 6 (Figure 4E); this value, although falling short of that of the GFP-rEag1 density, is more than 6-fold higher than that of the GFP-rEag2 density. When taken together, these findings suggest a potential correlation between the presence of the rEag1 post-CNBHD sequence and the punctate localization of the protein in question in 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)

Characterization of rEag1-I and rEag2-I chimeric channels. (A) Schematic representation of the construction of rEag1-I and rEag2-I chimeras. For all schematic cartoons hereafter, rEag1 and rEag2 sequences are shown in red and black, respectively. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated Eag constructs. Two-electrode voltage 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 +60 mV (rEag1) or from -100 to +40 mV (rEag2), with 10-mV increments. (C) Membrane localization of GFP-rEag1-I/rEag2-I channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of GFP-rEag1-I/rEag2-I channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for GFP-rEag1, GFP-rEag1-I, GFP-rEag2-I, and GFP-rEag2. The number in parenthesis denotes the amount of neurons analyzed. (*: 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 4: Characterization of rEag1-I and rEag2-I chimeric channels. (A) Schematic representation of the construction of rEag1-I and rEag2-I chimeras. For all schematic cartoons hereafter, rEag1 and rEag2 sequences are shown in red and black, respectively. (B) Representative K+ currents recorded from Xenopus oocytes over-expressing the indicated Eag constructs. Two-electrode voltage 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 +60 mV (rEag1) or from -100 to +40 mV (rEag2), with 10-mV increments. (C) Membrane localization of GFP-rEag1-I/rEag2-I channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of GFP-rEag1-I/rEag2-I channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for GFP-rEag1, GFP-rEag1-I, GFP-rEag2-I, and GFP-rEag2. The number in parenthesis denotes the amount of neurons analyzed. (*: significantly different from GFP-rEag1; t-test, p < 0.05)(#: significantly different from GFP-rEag2; t-test, p < 0.05)
Mentions: Both endogenous and over-expressed rEag1 channels displayed a punctate localization, which strongly suggests that one or more specific sequence motifs within its amino acid sequence may govern the subcellular distribution of the K+ channel in neurons. Although rEag1 and rEag2 share about 70% identity in amino acid sequence [4,6], a significant sequence divergence is present within the C-terminal post-CNBHD region, especially from residues A723 through S962 in rEag1 vs. L719 through F988 in rEag2 (Figure 3C). To test the hypothesis that a key structural domain within the post-CNBHD region may contribute to the punctate localization of rEag1 K+ channels, we generated two chimeric constructs (chimera rEag1-I and chimera rEag2-I) by exchanging the majority of the divergent post-CNBHD sequences between the two channel isoforms (Figure 4A). Upon over-expression in HEK293T cells or Xenopus oocytes, both of the chimeric channels yielded significant K+ currents and effective membrane surface expression (Figure 4B-C). We then over-expressed these GFP-tagged chimeras in hippocampal neurons in order to study their subcellular localization patterns. Confocal microscopic analysis revealed that the GFP-rEag1-I chimera displayed a rEag2-like pattern with only a few GFP puncta (Figure 4D) (see also Additional file 2). By way of contrast, the GFP fluorescence of the reverse chimera GFP-rEag2-I exhibited rEag1-like punctate localization (Figure 4D) (see also Additional file 3). We also quantified the puncta density of the GFP signal by calculating the number of GFP puncta per neuron. The GFP puncta densities of GFP-rEag1 and GFP-rEag2 were about 153 ± 5 and 17 ± 2, respectively (Figure 4E). This 9-fold difference in GFP puncta density between over-expressed GFP-rEag1 and GFP-rEag2 channels is almost equivalent to the previously measured 8-fold difference in puncta density between endogenous rEag1 and rEag2 proteins (see Figure 2B). The GFP puncta density of the GFP-rEag1-I chimera was dramatically reduced to about 31 ± 7, which is statistically similar to that of GFP-rEag2 (Figure 4E). In contrast, the GFP puncta density of the GFP-rEag2-I chimera was remarkably increased to about 109 ± 6 (Figure 4E); this value, although falling short of that of the GFP-rEag1 density, is more than 6-fold higher than that of the GFP-rEag2 density. When taken together, these findings suggest a potential correlation between the presence of the rEag1 post-CNBHD sequence and the punctate localization of the protein in question in 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