The punctate localization of rat Eag1 K+channels is conferred by the proximal post-CNBHD region
© Chuang et al.; licensee BioMed Central Ltd. 2014
Received: 8 September 2013
Accepted: 31 January 2014
Published: 4 February 2014
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.
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.
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.
KeywordsHippocampal neuron culture Subcellular localization Immunofluorescence Synaptosomal fractionation Electrophysiology
Voltage-gated potassium (K+) channels play various essential physiological roles in neurons, including controlling neuronal excitability, setting neuronal firing frequencies, shaping action potential waveforms, and modulating neurotransmitter release . The ether-à-go-go (Eag) K+ channel belongs to the EAG family of voltage-gated K+ channels that comprises three gene subfamilies; these are eag (KV10), erg (eag-related gene)(KV11), and elk (eag-like K+ channel)(KV12) . Results from in situ hybridization studies have indicated that Eag is neuron-specific K+ channel that is widely expressed in various regions of the brain [3–6]. In mammals, two Eag isoforms have been identified: Eag1 (KV10.1) and Eag2 (KV10.2) [4, 6–9].
Despite their abundant expression in the brain, the functional significance of Eag1 and Eag2 K+ channels remains obscure. One strategy to ascertain the neurophysiological role of voltage-gated ion channels is to identify their subcellular localization in neurons [10, 11]. Previous immunofluorescence characterization carried out in our laboratory has demonstrated that rat Eag1 (rEag1) and rat Eag2 (rEag2) K+ channels have different subcellular localizations over the dendrosomatic compartment in both hippocampal neurons and the retina [12, 13]; specifically, rEag1 channels exhibit a much broader range of expression, extending from somas to distal dendrites, and show a distinct punctate localization pattern. Since this punctate staining is co-localized with the presynaptic vesicle protein synaptophysin and the postsynaptic density protein densin-180 , it is likely that rEag1 is present within the synaptic region. Given that our previous immunofluorescence study was conducted with 14 days in vitro (DIV14) hippocampal cultures, wherein extensive neuronal connections are already formed, it was not possible to determine precisely whether rEag1 and/or rEag2 show axonal localization. A recent report applying immunofluorescence and quantum dot technology to DIV10 hippocampal neurons confirmed the punctate expression and synaptic localization of rEag1 channels ; moreover, this study also found that the immunofluorescence staining of rEag1 is co-localized with that of the axon marker tau, raising the possibility that rEag1 channels may be present in axons.
Emerging evidence suggests that specific sequence motifs within channel proteins may govern the subcellular distribution of ion channels in neurons [11, 15]. The proteins rEag1 and rEag2 share about 70% identity in amino acid sequence and thus belong to the same EAG K+ channel subfamily [4, 6]. Nevertheless, it remains unclear what constitutes the structural basis that dictates the differential subcellular localization of these two closely related voltage-gated K+ channels. In this study, we addressed this question by generating chimeras between rEag1 and rEag2 K+ channels. Quantitative analysis of these chimeras indicates that the proximal post-cyclic nucleotide-binding homology domain (post-CNBHD) region in the carboxyl (C) terminus confers the punctate localization of rEag1 K+ channels in hippocampal neurons.
The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA). The specificity of the anti-rEag1 and the anti-rEag2 antibodies has been previously verified .
Animals and hippocampal cultures
All procedures were in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2003) and approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University.
15-day pregnant Sprague–Dawley rats were purchased from the Facility for Animal Research of the National Yang-Ming University. Dissociated hippocampal cultures were prepared using a previously described protocol  with a minor modification . In brief, hippocampi were dissected from the brains of embryonic day 18 (E18) embryos, the brains of which were removed and placed in the Hank’s balanced salt solution that contains 10 mM HEPES (pH 7.4) and 1 mM sodium pyruvate. The hippocampus was dissected out and dissociated by incubation with 0.25% trypsin solution. The dissociated cells were plated on coverslips at a density of 200 and 1000 cells/mm2 for immunofluorescence and DNA transfection, respectively. Coverslips were coated with poly-D-lysine (1 mg/ml) (Sigma) and laminin (15 μg/ml) (Sigma). Cultures were maintained in the Neurobasal media supplemented with B27 (2%) and glutamax I (0.5 mM) (Invitrogen) in a humidified 5% CO2 incubator at 37°C.
Adult female Xenopus laevis (African Xenopus Facility) were anesthetized by immersion in ice water containing Tricaine (1.5 g/liter). Ovarian follicles were removed from Xenopus frogs, cut into small pieces, and incubated in the ND96 solution [(in mM) 96 NaCl, 2 KCl, 1.8 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.2]. To remove the follicular membrane, Xenopus oocytes were incubated in the Ca2+-free ND96 solution containing collagenase (2 mg/ml) on an orbital shaker (~200 rpm) for about 60-90 min at room temperature. After several washes with collagenase-free, Ca2+-free ND96, oocytes were transferred to ND96. Stage V-VI Xenopus oocytes were then selected for cRNA injection.
The cDNAs for rEag1 and rEag2 K+ channel subunits were kindly provided by Dr. Olaf Pongs (Institute fur Neurale, Signalverarbeitung, Zentrum fur Molekulare Neurobiologie). Green fluorescent protein (GFP)-tagged rEag1 and rEag2 constructs were made by subcloning the full length rEag1 and rEag2 cDNAs into the pEGFP mammalian expression vector (Clontech).
The design of the chimeras between rEag1 and rEag2 were based on sequence alignment. Chimeric channels were constructed by using the overlap PCR mutagenesis method. All constructs were verified by DNA sequencing (Genome Research Center, National Yang-Ming University).
For DNA transfection, human embryonic kidney (HEK) 293 T cells were maintained in DMEM (Invitrogen) supplemented with 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, and 10% (v/v) fetal bovine serum (Hyclone). For immunofluorescence and electrophysiology, cells were grown on poly-lysine-coated coverslips. After 24 hrs, HEK293T cells were transiently transfected with cDNAs by using the Lipofectamine 2000 (LF2000) reagent (Life Technologies).
Cultured hippocampal neurons at 7 days in vitro (DIV7) were also transfected by using LF2000. Briefly, various expression constructs were incubated with the LF2000 reagent for 20 min at room temperature. DNA-lipofectamine diluted in the complete medium was added to neuron culture wells. After 4-hr incubation at 37°C under 5% CO2, cells were washed gently three times with the culture media and maintained in the incubator before being examined under a fluorescence microscope.
For in vitro transcription, cDNAs were linearized with NotI. Capped cRNAs were transcribed in vitro from the linearized cDNA template with the mMessage mMachine T7 kit (Ambion). The apparent molecular weight and concentration of cRNAs were verified with gel electrophoresis and determined by spectrophotometry, respectively. For cRNA injection, the total volume of injection was always 41.4 nl per Xenopus oocyte. Injected oocytes were stored at 16°C in ND96.
Coverslips containing HEK293T cells or hippocampal neurons were rinsed in PBS [(in mM) 136 NaCl, 2.5 KCl, 1.5 KH2PO4, 6.5 Na2HPO4, pH 7.4] and then fixed with 4% paraformaldehyde in PBS at 4°C for 20 min. Cells were then permeabilized and blocked with a blocking buffer (5% normal goat serum in 20 mM phosphate buffer, pH 7.4, 0.1% (v/v) Triton X-100, and 0.45 M NaCl) for 60 min at 4°C. Appropriate dilutions of primary antibodies were applied in the blocking buffer overnight at 4°C. Immunoreactivities were visualized with goat-anti-mouse antibodies conjugated to Alexa568 or with goat anti-rabbit antibodies conjugated to Alexa488 (Molecular Probes). The fluorescence images were viewed and acquired with a Leica TCS SP5 laser-scanning confocal microscope.
Image analyses were performed with the ImageJ software (National Institute of Health). To determine the number of immunofluorescence clusters per fixed length of neurite, built-in “set scale” and “freehand tool” functions of the software were applied to trace multiple 100-μm neurite segments, followed by counting the number of PSD-95/rEag1/rEag2 puncta within each 100-μm neurite segment. Co-localization of PSD-95 (appearing as red punctate pixels) and rEag1/rEag2 (appearing as green punctate pixels) puncta within each 100-μm neurite segment was recognized by identifying the presence of overlapping punctate pixels. For neurons transfected with various GFP-tagged constructs, the number of GFP puncta per neuron was also estimated using ImageJ. Statistical analyses were executed with the Origin 7.0 software (Microcal Software). All numerical data are shown as mean ± standard error (SEM).
Subcellular fractionation of rat brain and preparation of PSDs
Subcellular and PSD fractions of adult rat brains were prepared as described previously . In brief, adult rat forebrains were homogenized in the buffer H1 [(in mM) 320 sucrose, 1 NaHCO3, 0.5 CaCl2, 0.1 PMSF] containing a cocktail of protease inhibitors (Roche) and centrifuged at 1,400×g to remove nuclei and other large debris (P1). The S1 fraction was subject to centrifugation at 13,800xg to obtain the crude synaptosome fraction (P2). The pellet was resuspended in the buffer H2 [(in mM) 0.32 M sucrose and 1 mM NaHCO3)] and layered onto the top of the discontinuous sucrose density gradient by using 0.85, 1.0, and 1.2 M sucrose layers. The gradient was centrifuged at 65,000xg for 2 hrs in a Beckman Instruments SW-28 rotor and the synaptosomal fraction (SPM) was recovered from the 1.0-1.2 M sucrose interface. The synaptosomal fraction was extracted in ice-cold 0.5% Triton X-100/50 mM Tris–HCl (pH 7.9) for 15 min and centrifuged at 32,000xg for 45 min to obtain the PSD I pellet. The pellet was resuspended and further extracted a second time with 0.5% Triton X-100/50 mM Tris–HCl (pH 7.9), followed by centrifugation at 200,000×g for 45 min to obtain the PSD II pellet. Protein concentration was determined by the BCA protein assay kit (Thermo). For immunoblotting, 25 μg (H, S1, P2, and SPM) or 5 μg (SPM, PSD I, and PSD II) of proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes, incubated with the primary antibodies, and imaged with the enhanced chemiluminescence method (Thermo).
For HEK293T cells, conventional whole-cell patch clamp technique was used to record Eag K+ currents as described previously . In brief, recordings were performed at 24-48 hrs post-transfection. Patch electrodes with a resistance of ~4 MΩ were pulled on a Narishige PP-830 electrode puller and were filled with a solution containing (in mM) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. External bath solution comprised (in mM) 140 NaCl, 5 KCl, 1 CaCl2, and 10 HEPES, pH 7.2.
Conventional two-electrode voltage clamp recording in Xenopus oocytes were performed as described previously . In brief, 2-3 days after cRNA injection, oocytes were functionally assayed in a recording bath containing about ~200 μl of the Ringer solution [(in mM): 115 NaCl, 3 KCl, 1.8 CaCl2, 10 HEPES, pH 7.2]. An agarose bridge was used to connect the bath solution with a ground chamber (containing 3 M KCl) into which two ground electrodes were inserted. Borosilicate electrodes (0.1 –1 MΩ) used in voltage recording and current injection were filled with 3 M KCl.
Voltage-clamp protocols were applied with the pCLAMP 8.2/9.0 software (Molecular Devices). Data were acquired with an Axopatch 200A amplifier (Molecular Devices) (for HEK293T cells) or OC-725C oocyte clamp (Warner) (for Xenopus oocytes), followed by digitization at 10 kHz with the Digidata 1320A/1322A system (Molecular Devices). Also by using the pCLAMP 8.2/9.0 software, data were filtered at 1 kHz and passive membrane properties were compensated with the -P/4 leak subtraction method. All recordings were performed at room temperature (20-22°C).
Cells with large currents in which voltage clamp errors might appear were excluded from data analyses. Kinetic fitting of Eag K+ current traces were implemented with the pCLAMP 8.2/9.0 software. Subsequent numerical analyses and data plotting were performed with the Origin 7.0 software. All numerical data are shown as mean ± SEM.
Different localizations of rEag1 and rEag2 channels in axons and synapses
We also found that rEag2 was co-localized with MAP2 in all three populations of cultured hippocampal neurons (Figure 1C). In addition, we found that a fraction of the immunofluorescence signal of rEag2 was co-localized with that of the tau immunofluorescence signal, especially in immature DIV3 neurons (Figure 1D). In contrast to rEag1, which was found to be universally present throughout the axonal compartment, in DIV7 and DIV12 neurons, rEag2 seemed to display a pattern in axons that was relatively restricted and showed a low overall level of expression (Figure 1D). Most importantly, in virtually all the neuron samples we analyzed, rEag2 did not show a significant punctate distribution within either the dendrosomatic or the axonal compartments.
An alternative approach to addressing the synaptic localization of proteins is to examine their subcellular fractionation. By this approach, the differential localization of synapse-related proteins can be demonstrated via their distinct enrichment patterns in the synaptosomal (SPM) and the two PSD (PSD I and PSD II) fractions. As depicted in Figure 2D, the presynaptic marker synaptophysin was highly enriched in the SPM fraction and was virtually absent in the PSD II fraction. In contrast, PSD-95, as well as β-actin, was found to be highly enriched in both the PSD I and the PSD II fractions. Moreover, Figure 2D clearly demonstrates that rEag1 protein was significantly enriched in all three of the synaptosomal sub-fractions. Taken together, these findings imply that a significant number of rEag1 K+ channels may be present at presynaptic axonal terminals and/or on the postsynaptic dendritic spines.
Differential subcellular localization of GFP-tagged rEag1 and rEag2 channels in hippocampal neurons
Characterization of the subcellular localization of chimeric channels in hippocampal neurons
The voltage-dependent gating properties of the chimeric channels
Steady-state voltage-dependent activation parameters of the rEag1 and rEag2 chimeric channels
-17.5 ± 0.9
19.9 ± 1.5
-19.2 ± 0.8
17.8 ± 1.2
-8.5 ± 0.7*
24.6 ± 0.7*
-16.1 ± 0.6
14.3 ± 1.5*
-15.8 ± 1.3
18.3 ± 1.0
-43.6 ± 1.2
18.3 ± 1.1
-40.0 ± 0.7
17.8 ± 0.9
-42.6 ± 0.5
19.0 ± 0.7
-54.1 ± 1.8*
19.1 ± 1.4
-44.8 ± 1.4
18.5 ± 1.3
In this report, we began by inspecting the subcellular localization of rEag1 and rEag2 K+ channels in young and mature neurons in culture. Our immunofluorescence analysis indicated that, in addition to the dendrosomatic domain, both two Eag isoforms were present in axons of hippocampal neurons. Most importantly, significant punctate distribution was observed in both the dendrosomatic and the axonal compartments for rEag1 channels only. In addition, by over-expressing GFP-tagged constructs, we found that GFP-rEag1, but not GFP-rEag2, displayed significant punctate localization in hippocampal neurons, which is reminiscent of the differential subcellular localization of endogenous rEag1 and rEag2 channels. We further addressed the structural basis underlying the differences in localization patterns and demonstrated that the proximal post-CNBHD region (rEag1 A723-R807) in the C-terminus confers the punctate localization of rEag1 K+ channels in hippocampal neurons. To the best of our knowledge, this is the first direct evidence showing that the post-CNBHD region seems to contribute to the subcellular localization of Eag K+ channels.
The assessment of protein expression in axons may be hindered by the presence of extensive neurite connections within cultured neurons, as was seen in our previous study of DIV14 hippocampal cultures . In this study we focused on the immunofluorescence characterization of younger neurons where the neurite network is less sophisticated. By closely inspecting DIV3 neurons, wherein one fast-growing neurite becomes the axon and the other slow-growing neurites become the dendrites , we observed significant immunofluorescence signal in MAP2-negative, tau-positive neurites for both rEag1 and rEag2 K+ channels. Similar results were also found in DIV7 neurons, wherein dendritic spine formation and synaptic connections can be clearly identified . Together with our previous demonstration of their dendrosomatic localization , we propose that in hippocampal neurons the two rat Eag isoforms seem to play distinct but essential physiological roles in modulating dendrosomatic excitability, as well as in the propagation of action potential in axons. The foregoing inference is reminiscent of the physiological significance of the prototypic Eag channel that was cloned from Drosophila. Electrophysiological recordings from motor neurons in Drosophila with mutations in the eag gene revealed an increase in spontaneous neuronal firing and presynaptic transmitter release , which is consistent with the somatic and axonal localization of Drosophila Eag K+ channels. Moreover, Drosophila with mutations in the eag gene were found to be deficient in antennal sensitivity to a subset of odorants , which suggests that Eag K+ channels may also display dendritic localization in the olfactory receptor neurons of Drosophila antennae.
In clear agreement with the conclusion drawn from our previous immunofluorescence studies [12, 13], the current study demonstrates that rEag1, but not rEag2, displays significant punctate localization in both the dendrosomatic and the axonal compartments of mature hippocampal neurons. A significant fraction of rEag1 puncta was found to be co-localized with synaptic markers such as synaptophysin , densin-180 , and PSD-95 (Figure 2A). Furthermore, fractionation analysis revealed that rEag1 was highly enriched in the synaptosomal fraction (Figure 2B). We therefore propose that rEag1 channels are significantly expressed at presynaptic axonal terminals and on postsynaptic dendritic spines, and may play a critical role in controlling neurotransmitter release and postsynaptic signaling.
Specific structural domains have been identified to explain the structure-function mechanisms underlying the divergent voltage-gating processes of different K+ channels [23, 24]. Similarly, various sequence motifs within different voltage-gated K+ channels have been shown to govern their subcellular localization and the targeting of channel proteins to different neuronal compartments [11, 15]. Despite the presence of about 70% identity in amino acid sequence between the Eag1 and Eag2 K+ channel proteins [4, 6], the structural bases of their different voltage-gating properties and subcellular localizations have remained largely elusive. Previous biophysical analysis of a series of different chimeras between human Eag1 and Eag2, for example, revealed that the transmembrane regions alone were not sufficient to explain the differences in their gating kinetics and steady-state voltage-dependence [25, 26]. In addition, similar to our results here (Figure 8), non-membrane regions per se were found not to determine their gating behaviors . Together these results suggest that the divergent voltage-gating property between the two Eag isoforms may rather arise from interactions among multiple structural domains within the channel protein.
In this study we found that the GFP-tagged rEag1 chimeras (1-I and 1-II) that harbor the proximal post-CNBHD region of rEag2 displayed a dramatic reduction in hippocampal neuron fluorescence puncta (Figures 4E and 5E). Conversely, notable punctate patterns were observed with the GFP-tagged rEag2 chimeras (2-I and 2-II) which contain the proximal post-CNBHD region of rEag1 (Figures 4E and 6E). Finally, the rEag1 truncation mutant K848X that lacks the distal post-CNBHD region still displayed significant punctate localization in hippocampal neurons (Figure 7). Taken these findings as a whole, they strongly support the hypothesis that the punctate localization of rEag1 K+ channels is conferred by the proximal post-CNBHD region. However, it remains to be determined whether this region alone is sufficient to determine the pre/post-synaptic localization of rEag1. One alternative is that the synaptic targeting of rEag1 channels may involve interactions between a subset of the proximal post-CNBHD sequences and other protein domains. Furthermore, we cannot rule out the possibility that certain proximal-post-CNBHD-interacting protein(s) may be required for the punctate/synaptic localization of rEag1 protein.
As mentioned in the Results section, for the K848X truncation mutant and the chimeras that do display prominent punctate localization, their GFP puncta densities are about 5-fold to 7-fold higher than that of wild-type rEag2. Nevertheless, the GFP puncta densities of these constructs are still actually lower than that of wild-type rEag1. The reason for this discrepancy is unknown. One possible explanation is that in neurons, the chimeric and mutant constructs are less effective in terms of protein expression, membrane trafficking, and/or puncta formation.
Immunofluorescence studies reveal that in hippocampal neurons, rEag1 and rEag2 K+ channels are present in both the dendrosomatic and the axonal compartments. In addition, rEag1 protein is significantly expressed within synaptic regions and displays a distinct punctate localization pattern. By studying a series of different chimeric constructs between rEag1 and rEag2, we have determined that the proximal post-CNBHD region of the rEag1 protein confers punctate localization of rEag1 K+ channels. These findings highlight a new direction for studies in this area and provide important insights that should help the elucidation of the physiological significance of Eag K+ channels in the brain.
Carboxyl assembly domain
Cyclic nucleotide-binding homology domain
Days in vitro
Green fluorescent protein
Microtubule-associated protein 2
We thank Dr. Chih-Yung Tang for technical guidance and critically reading the manuscript. This work was supported by research grants from National Science Council (NSC101-2320-B-010-038-MY3) and from the Aim for the Top University Plan, Ministry of Education, Taiwan.
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