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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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The biophysical properties of the chimeric Eag channels. Comparison of the voltage-dependent gating properties of the rEag1 (A) or rEag2 (B) chimeras with their wild-type (WT) counterparts. Steady-state voltage dependence (activation curve) is illustrated as the fraction of open channels (Po) against the corresponding membrane potential. Activation time constants at indicated potentials were obtained from single exponential fits to the late rising phase of Eag K+ currents. Deactivation time constants were derived from single exponential fits to the decay phase of Eag K+ currents at the indicated tail potential in response to a +40 mV test pulse. All values are presented as mean ± SEM. Data were collected and analyzed as described previously [18].
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Figure 8: The biophysical properties of the chimeric Eag channels. Comparison of the voltage-dependent gating properties of the rEag1 (A) or rEag2 (B) chimeras with their wild-type (WT) counterparts. Steady-state voltage dependence (activation curve) is illustrated as the fraction of open channels (Po) against the corresponding membrane potential. Activation time constants at indicated potentials were obtained from single exponential fits to the late rising phase of Eag K+ currents. Deactivation time constants were derived from single exponential fits to the decay phase of Eag K+ currents at the indicated tail potential in response to a +40 mV test pulse. All values are presented as mean ± SEM. Data were collected and analyzed as described previously [18].

Mentions: In addition to divergent subcellular localization patterns, rEag1 and rEag2 channels also have different gating properties including steady-state voltage dependence and activation/deactivation kinetics [4-6]. A similar disparity has also been observed in human Eag1 and Eag2 channels [7,9]. To understand whether sequence divergence in the post-CNBHD region may also contribute to the distinct biophysical properties of the two Eag K+ channel isoforms, we went on to analyze the gating property of the chimeras. The left panels in Figure 8, as well as Table 1, demonstrate that the steady-state voltage dependence (PO-V curve) properties of the chimeras are similar to those of their wild-type counterparts, indicating that sequence divergence in the post-CNBHD region is not able to account for the ~40-mV discrepancy in voltage activation between rEag1 and rEag2. Furthermore, despite about two-fold difference in the activation kinetics between the two Eag isoforms, exchanging post-CNBHD sequences led to only a small acceleration in the activation kinetics of all chimeras (Figure 8, middle panels). Moreover, the introduction of chimeric post-CNBHD sequences did not have any significant effect on the deactivation kinetics of the two Eag isoforms (Figure 8, right panels). Taken together, our biophysical findings demonstrate that the sequence differences in the post-CNBHD region are unable to explain the divergence in the voltage-dependent gating properties of rEag1 and rEag2 K+ 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)

The biophysical properties of the chimeric Eag channels. Comparison of the voltage-dependent gating properties of the rEag1 (A) or rEag2 (B) chimeras with their wild-type (WT) counterparts. Steady-state voltage dependence (activation curve) is illustrated as the fraction of open channels (Po) against the corresponding membrane potential. Activation time constants at indicated potentials were obtained from single exponential fits to the late rising phase of Eag K+ currents. Deactivation time constants were derived from single exponential fits to the decay phase of Eag K+ currents at the indicated tail potential in response to a +40 mV test pulse. All values are presented as mean ± SEM. Data were collected and analyzed as described previously [18].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: The biophysical properties of the chimeric Eag channels. Comparison of the voltage-dependent gating properties of the rEag1 (A) or rEag2 (B) chimeras with their wild-type (WT) counterparts. Steady-state voltage dependence (activation curve) is illustrated as the fraction of open channels (Po) against the corresponding membrane potential. Activation time constants at indicated potentials were obtained from single exponential fits to the late rising phase of Eag K+ currents. Deactivation time constants were derived from single exponential fits to the decay phase of Eag K+ currents at the indicated tail potential in response to a +40 mV test pulse. All values are presented as mean ± SEM. Data were collected and analyzed as described previously [18].
Mentions: In addition to divergent subcellular localization patterns, rEag1 and rEag2 channels also have different gating properties including steady-state voltage dependence and activation/deactivation kinetics [4-6]. A similar disparity has also been observed in human Eag1 and Eag2 channels [7,9]. To understand whether sequence divergence in the post-CNBHD region may also contribute to the distinct biophysical properties of the two Eag K+ channel isoforms, we went on to analyze the gating property of the chimeras. The left panels in Figure 8, as well as Table 1, demonstrate that the steady-state voltage dependence (PO-V curve) properties of the chimeras are similar to those of their wild-type counterparts, indicating that sequence divergence in the post-CNBHD region is not able to account for the ~40-mV discrepancy in voltage activation between rEag1 and rEag2. Furthermore, despite about two-fold difference in the activation kinetics between the two Eag isoforms, exchanging post-CNBHD sequences led to only a small acceleration in the activation kinetics of all chimeras (Figure 8, middle panels). Moreover, the introduction of chimeric post-CNBHD sequences did not have any significant effect on the deactivation kinetics of the two Eag isoforms (Figure 8, right panels). Taken together, our biophysical findings demonstrate that the sequence differences in the post-CNBHD region are unable to explain the divergence in the voltage-dependent gating properties of rEag1 and rEag2 K+ 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