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First published online 20 February 2007
doi: 10.1242/jcs.03396

Journal of Cell Science 120, 953-963 (2007)
Published by The Company of Biologists 2007
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Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment

Hanne B. Rasmussen1,*, Christian Frøkjær-Jensen1, Camilla S. Jensen1, Henrik S. Jensen1, Nanna K. Jørgensen1, Hiroaki Misonou2,3, James S. Trimmer2, Søren-Peter Olesen1 and Nicole Schmitt1

1 Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
2 Department of Pharmacology, School of Medicine, University of California, Davis, CA 95616, USA
3 Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA


Figure 1
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  		Fig. 1. Co-immunolocalization of KCNQ2 and KCNQ3 with ankyrin-G in CA1 of rat hippocampus. (A-F) Cross-section of rat hippocampal CA1 double-labeled for KCNQ2 (A,D) and ankyrin-G (B,E). (G-L) Cross-section of rat hippocampal CA1 double-labeled for KCNQ3 (G,J) and ankyrin-G (H,K). KCNQ2 and KCNQ3 both co-localize with ankyrin-G in this region. Panels D-F and J-L represent a magnified view of the pyramidal cell layer. ol, oriens layer; py, pyramidal cell layer; rad, stratum radiatum.

 

Figure 2
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  		Fig. 2. Endogenous expression of KCNQ channel subunits in primary hippocampal neurons. (A) Reverse-transcription PCR analysis from cultured rat hippocampal neurons. RT-PCR was performed using 0.15 µg of DNase I-treated, total RNA from cultured hippocampal neurons (10 DIV). KCNQ2 (Q2), KCNQ3 (Q3), KCNQ4 (Q4) and KCNQ5 (Q5) were detected in the cultures. M, Marker; Q1, KCNQ1. (B) Immunoblot analysis of endogenous expression of KCNQ2 and KCNQ3 in cultured hippocampal neurons. Rat brain membranes (RBM) and lysates of primary hippocampal neurons (HN, 10 DIV) were subjected to western blotting and examined for KCNQ2 and KCNQ3 expression. Stars indicate bands that correspond to KCNQ2 and KCNQ3, respectively. Both channel subunits were detected in the hippocampal cultures. (C) Immunodetection of endogenous KCNQ2 and KCNQ3 in the hippocampal cultures. Hippocampal neurons (21 DIV) were double-labeled for endogenous KCNQ2 and MAP2 or endogenous KCNQ3 and MAP2. Both KCNQ2 and KCNQ3 displayed intracellular, somatic localization as well as localization to the AIS (MAP2-negative). Bar, 20 µm. Arrows mark the position of the AIS, while arrowheads point to the distal axon. (Right) Analysis of the immunofluorescence intensity profiles of endogenous KCNQ2 and endogenous KCNQ3 along the main axon up to 100 µm from the soma from the two images demonstrated that the two subunits were enriched in the AIS compared to the distal axon. (D) Electrophysiological detection of endogenous KCNQ subunits. Shown are currents recorded from a cultured neuron (10 DIV) in the absence (control) and presence of the KCNQ-specific blocker XE-991. Whole-cell currents were recorded from voltage-clamped neurons, and a standard hyperpolarizing protocol (top traces) was used to measure the M-current. Cells were held at –70 mV and then depolarized to –20 mV before they were stepped back to hyperpolarized voltages to measure the M-current. Difference currents were obtained by subtracting the control trace at –40 mV from that in the presence of XE-991. Below is shown a time series of a slowly activating current at –40 mV recorded every 5 seconds in the absence or presence of XE-991. The current is stable in the whole cell recording configuration and is partially blocked by 10 µM XE-991. (E) Immunoblot detection of KCNQ2 expression in KCNQ2-transfected COS-1 cells (COS-1 Q2), rat brain membranes (RBM), primary hippocampal neurons (HN) and KCNQ2-transfected primary hippocampal neurons (HN-Q2). Note the high level of exogenous KCNQ2 expression compared with the level of endogenous KCNQ2.

 

Figure 3
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  		Fig. 3. Exogenously expressed hKCNQ2-cmyc and hKCNQ3-FLAG are primarily localized intracellulary in primary hippocampal neurons. Hippocampal neurons expressing hKCNQ2-cmyc (hQ2-myc) or hKCNQ3-FLAG (hQ3). Transfected neurons were fixed and double-labeled with anti-cmyc or anti-hKCNQ3 together with anti-ankyrin-G (AnkG) (A), anti-MAP2 (B) or anti-protein disulfide isomerase (PDI) (C) antibodies. Neither hKCNQ2-cmyc nor hKCNQ3-FLAG displayed a selective enrichment at the AIS (A) and no accumulation of the channel subunits in MAP-2 negative compartments was observed (B). Instead, both subunits displayed a primarily intracellular localization that partly co-localized with the endoplasmic reticulum marker PDI (C). White boxes in the merged images in C represent the magnified regions displayed below. Arrows mark the position of the AIS, while arrowheads point to the distal axon. Bar, 20 µm.

 

Figure 4
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  		Fig. 4. Co-expressed hKCNQ2-cmyc and hKCNQ3-FLAG are localized to the AIS of primary hippocampal neurons. Hippocampal neurons were co-transfected with hKCNQ2-cmyc and hKCNQ3-FLAG, fixed and triple-labeled for hKCNQ2-cmyc, hKCNQ3-FLAG and either ankyrin-G (A) or MAP2 (B). Bar, 20 µm. hKCNQ2-cmyc and hKCNQ3-FLAG labeling was enriched in the AIS, which is positive for ankyrin-G, but devoid of MAP2. Additional staining could also be observed intracellularly as well as in the more distal parts of the axon. Arrows mark the position of the AIS, while arrowheads point to the distal axon. (C) Representative current traces of an hKCNQ2-cmyc/hKCNQ3-FLAG transfected neuron in the absence or presence of 10 µM XE-991. Protocols were as described in Fig. 2. A large, slowly non-inactivating M-current could be measured in transfected neurons. For comparison with the endogenous M-current, the bottom trace shows a time-series of a transfected neuron (Q2+Q3) and a GFP-transfected neuron (Control) before and after application of XE-991 (10 µM). The level of XE-991-sensitive current is on average increased 15-fold in neurons transfected with hKCNQ2-cmyc/hKCNQ3-FLAG compared with that in controls (see Fig. 4B for quantification).

 

Figure 5
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  		Fig. 5. Overview of AIS localization and current levels for the various KCNQ constructs. (A) Summary of observed KCNQ AIS localization in neurons transfected with the listed combinations of constructs. AIS/distal axon (AIS/DA) and AIS/dendrite (AIS/den) ratios were determined for each combination of constructs for 29-30 cells in at least three different cultures. Q2+Q3{Delta} was statistically different from wild-type Q2+Q3 for both KCNQ2 and KCNQ3 immunofluorescence quantifications, whereas Q2{Delta}+Q3 was not. In addition, Q2+Q3{Delta} was statistically different from Q2{Delta}+Q3{Delta} in the KCNQ2 quantification, but not for the KCNQ3 quantification. One-way ANOVA and Bonferroni's post-test were used in the comparison of Q2+Q3 vs Q2{Delta}+Q3, Q2+Q3 vs Q2+Q3{Delta} and Q2{Delta}+Q3 vs Q2+Q3{Delta} (*P<0.05, **P<0.001). (B) Summary of current density levels observed in neurons transfected with the listed constructs. All neurons were co-transfected with GFP. Recordings were carried out on cultures from at least two different days. Q2+Q3 and {Delta}Q2+{Delta}Q3 were statistically different from controls. One-way ANOVA and Dunnet's multiple comparison post-test were used to compare the various constructs with the control (*P<0.05). (A+B) Data are displayed as mean±standard error of mean. Abbreviations: control, GFP; Q2, hKCNQ2-cmyc; Q3, hKCNQ3-FLAG; Q2{Delta}, hKCNQ2-delAnkG; Q3{Delta}, hKCNQ3-delAnkG; Q2A, Q2-AAA; and Q3-A, Q3-AAA.

 

Figure 6
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  		Fig. 6. The C-termini of KCNQ2 and KCNQ3 contain a potential ankyrin-G-interaction domain. (A) Schematic structure of the KCNQ2 and KCNQ3 subunits with indications of domains of protein-protein interaction. Indicated is also the presence of the ankyrin-G-interaction site in the C-termini of both the KCNQ2 and KCNQ3 subunits. Each circle represents an amino acid. Abbreviations: AKAP, A-kinase anchoring protein, CaM; calmodulin; si, subunit interaction. (B) Clustal W alignment of the 9 amino acid ankyrin-G-binding motif of Nav1.6 along with the ankyrin-G-interaction domain of KCNQ2 and KCNQ3, the corresponding accession no. given in brackets. Red indicates small, hydrophobic residues, blue represents acidic residues, and pink labels basic residues, while green is representative of amino acids containing hydroxyl or amine sidegroups. Asterisks (*) indicate that the residues in that column are identical in all sequences of the alignment; colons (:) indicate that conserved substitutions have been observed.

 

Figure 7
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  		Fig. 7. The ankyrin-G-interaction domain mediates retention of KCNQ2/3 complexes at the AIS. (A) Hippocampal neurons were co-transfected with hKCNQ2-delAnkG ({Delta}803-844) (hQ2-delAnkG) + hKCNQ3-FLAG (hQ3), hKCNQ2-cmyc (hQ2-cmyc) + hKCNQ3-delAnkG ({Delta}826-872) (hQ3-delAnkG), hKCNQ2-delAnkG + hKCNQ3-delAnkG, or KCNQ2-E810A/S811A/D812A (KCNQ2-AAA) and KCNQ3-E837A/T838A/D839A (KCNQ3-AAA). The cells were fixed and triple-labeled for hKCNQ2, hKCNQ3 and ankyrin-G. Bar, 20 µm. hKCNQ2-delAnkG/hKCNQ3-FLAG were enriched at the AIS to the same extent as wild-type hKCNQ2/hKCNQ3. For hKCNQ2-cmyc/hKCNQ3-delAnkG complexes a significant reduction in the AIS enrichment was observed (see also Fig. 5). For hKCNQ2-delAnkG/hKCNQ3-delAnkG and hKCNQ2-AAA/hKCNQ3-AAA complexes AIS enrichment was abolished. (B) Representative current traces of a hKCNQ2-delAnkG/hKCNQ3-delAnkG transfected neuron in the absence or presence of 10 µM XE-991. Protocols were as described in Fig. 2. Current levels were comparable with those observed for full-length KCNQ2-cmyc/KCNQ3-FLAG transfected neurons, thus the hKCNQ2-delAnkG/hKCNQ3-delAnkG complex is still trafficked to the cell surface.

 

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© The Company of Biologists Ltd 2007